Physiological characterisation of a pH- and calcium-dependent sodium uptake mechanism in the freshwater crustacean, Daphnia magna
Department of Biology, McMaster University, Hamilton, Ontario, Canada
* Author for correspondence at National Institute for Nutrition and Seafood Research, Postboks 2029 Nordnes, 5817 Bergen, Norway (e-mail: chris.glover{at}nifes.no)
Accepted 1 December 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: acid precipitation, soft water, hardness, osmoregulation, invertebrate, Daphnia magna
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanism of sodium uptake in freshwater organisms has been extensively
investigated. Sodium transport across the gill of freshwater-adapted crabs is
thought to be powered by active proton extrusion (for a review, see
Kirschner, 2004). An apical
V-type H+-ATPase provides an electrochemical gradient for the
passage of sodium ions from freshwater into the gill cell via a
sodium channel. Sodium is consequently driven across the basolateral surface
by the ATP-dependent sodiumpotassium pump
(Na+/K+-ATPase). This is similar to the mechanism of
uptake in freshwater fish (Evans et al.,
1999
). Conversely in freshwater-adapted euryhaline crayfish,
apical sodium transfer is likely achieved by an apical sodiumproton
exchange mechanism, where the extrusion of protons and the uptake of sodium
are mediated by the same transport moiety
(Kirschner, 2004
). In the
freshwater cladoceran Daphnia magna it is known that sodium uptake is
saturable, indicating a specific transport mechanism is involved (e.g.
Stobbart et al., 1977
;
Potts and Fryer, 1979
;
Glover et al., 2005
).
Furthermore sodium uptake is reduced as pH is decreased, suggesting that
sodium uptake may be linked to proton excretion
(Potts and Fryer, 1979
).
Proton-linked sodium uptake likely explains the high sensitivity of
freshwater animals to aquatic acidification. Fish, molluscs and crustaceans
have disappeared from many fresh waters as a result of acid precipitation
(e.g. Leivestad et al., 1976).
The mechanism behind such mortalities appears to be the breakdown in sodium
ion regulation (see Vangenechten et al.,
1989
; Wood, 1989
).
In freshwater fish, mortality in acid waters appears to be mediated by an
inhibition of sodium influx, and an enhanced sodium efflux
(Wood, 1989
). The influx
inhibition is likely a consequence of acid interference with sodium transport
processes, be it a direct competition between protons and sodium ions for
uptake (Wood, 1989
), or an
indirect effect caused by the loss of the outward proton gradient that drives
inward sodium flux (Lin and Randall,
1995
). In fish, the presence of calcium in acid waters appears to
protect against sodium depletion. Raising calcium levels in laboratory
experiments is believed to replace the calcium leached from tight junctions by
enhanced acidity (see Wood,
1989
). This calcium addition reduces junction permeability,
decreases paracellular sodium efflux and favourably influences whole body
sodium status.
Mechanistic knowledge of sodium transport pathways in the highly sensitive
freshwater crustacean Daphnia magna will contribute greatly to our
understanding of how these organisms respond to environmental stressors such
as acid precipitation, and also to environmental metal contamination. Silver,
for example, inhibits sodium uptake pathways and thus causes mortality at
extremely low concentrations (Bianchini and
Wood, 2003). The inability to replace lost sodium rapidly depletes
whole body sodium concentrations and results in mortality. Acute median lethal
toxicity values of less than 1 µg l1, make daphnids the
most sensitive of all freshwater animals to environmental silver (for a
review, see Wood et al.,
2002
). Mechanistic knowledge of sodium uptake pathways would
enhance our understanding of the mode of toxicity of silver and other metal
toxicants that are likely to interfere with this process (e.g. copper).
In this study a transport kinetic approach has been utilised to determine
the effects of hydrogen and calcium ions on sodium influx in Daphnia
magna. This type of approach is beneficial in that changes in parameters
derived from MichaelisMenten analysis may provide mechanistic
information. Alterations in transport affinity and/or transport capacity may
be characteristic of either competitive or non-competitive interactions at the
transport site, or a combination of both
(Cornish-Bowden, 1979). The
nature of acid and calcium interactions with sodium influx will provide
insight into mechanisms of sodium influx, and will have implications for the
response of freshwater crustaceans to anthropogenic modifications of the
natural environment, including metal pollution and acid precipitation.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Experimental manipulations
The effect of water calcium and pH on Daphnia sodium influx was
determined at three calcium concentrations (0, 0.5 and 1 mmol
l1; as CaSO4), and at three pH levels (4, 6 and
8). Experimental media were all prepared from deionised water (>17.5
M cm; Barnstead Nanopure II, Dubuque, IA, USA). To permit kinetic
analysis of sodium uptake at each of these nine water chemistries, five sodium
concentrations (50, 150, 300, 750 and 1500 µmol l1; as
NaCl) were analysed. This resulted in a total of 45 experimental chambers (100
ml of solution in an acid-washed 250 ml glass beaker; Pyrex). To each chamber
22Na (
1 kBq ml1 as NaCl; Perkin Elmer,
Boston, MA, USA) was added as a marker of sodium influx. Adjustment of pH (0.1
N KOH or 0.1 N HNO3) was performed
16 h prior to experiment
commencement, with a final adjustment of pH within 3 h of daphnid
introduction. Six Daphnia were added to each chamber, and influx was
monitored over 1 h. The high solution to biomass ratio, the use of
experimental water reconstituted from deionised water, and the short flux
measurement duration sought to minimise the contribution of organic carbon,
which could potentially complicate sodium metabolism
(Glover et al., 2005
).
To further delineate the actions of calcium on sodium influx kinetics, a
follow-up study utilising a similar protocol was employed. The calcium
concentration-dependence of sodium uptake was examined over a wide range of
calcium concentrations (0, 50, 100, 500, 1000, 5000 µmol
l1; as calcium gluconate), in the presence of low (50
µmol l1) or high (1000 µmol l1)
sodium water concentrations. This experiment was conducted with two sodium
salts: sodium chloride and sodium gluconate. The use of the gluconate salt
introduced sodium into solution with an impermeant anion, and thus permitted
an additional analysis of the influence of Cl on sodium
influx. These experiments used identical radiotracer specific activities
(1 kBq ml1 as NaCl; Perkin Elmer), water volumes (100
ml), daphnid numbers (6), and influx times (1 h), to that described above,
with a pH
6.
The effect of amiloride
(N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride;
Sigma, St Louis, MO, USA) on sodium influx was examined at two sodium
concentrations (50 µmol l1 and 300 µmol
l1 as NaCl). Amiloride (10, 50, 100, 500, 1000, 5000 or 10
000 µmol l1) was added from a concentrated stock solution
to 50 ml of an appropriate sodium solution. Five Daphnia were added
to each test chamber, and influx was determined from uptake of radiotracer
(22Na; 1 kBq ml1 as NaCl; Perkin Elmer) over
15 min. This time was chosen to minimise the acutely toxic effects of the
amiloride exposure. In an additional treatment, daphnids were pre-exposed to
the highest amiloride concentration (10 000 µmol l1) for
15 min, rinsed in synthetic Lake Ontario water for
1 min, then added to
amiloride-free experimental chambers containing radiolabelled sodium for 15
min.
Sodium influx determination and whole body sodium measurement
Daphnia from all experimental treatments were analysed for sodium
influx in an identical manner. Following removal from experimental chambers,
daphnids were rinsed (10 s) in a high sodium displacement solution
(
1 mol l1 NaCl), with two subsequent rinses (
15 s
each) in deionised water. Animals were blotted dry, weighed (UMT2,
Mettler-Toledo, Greifensee, Switzerland; 0.001 mg precision), and counted for
-activity (Canberra-Packard, Minaxi Auto-gamma 5000, Meridian, CT,
USA). Sodium influx was calculated from the equation
Jin=c.p.m./(SAmt), where c.p.m. is the
counts per minute in the daphnid, SA is the specific activity of the
exposure water (c.p.m. µequiv1), m represents
the daphnid wet mass (in g, corrected for trapped carapace water by
multiplying by 1.25; Stobbart et al.,
1977
), and t is the time of exposure in h. This resulted
in a sodium influx expressed as µequiv g wet mass h1.
Daphnids from the combined pH/calcium experimental protocol were also analysed for whole body sodium content. Individual animals were digested in 50 µl of concentrated H2SO4 (trace metal grade; Fisher, Nepean, ON, Canada), before being diluted with deionised water to an appropriate concentration for analysis via flame atomic absorption spectrophotometry (220FS, Varian, Palo Alto, CA, USA). Whole body sodium concentrations were calculated as the sodium concentration in the daphnid, per unit wet mass, again accounting for trapped carapace water.
Data analysis
Data points have been routinely expressed as means ±
S.E.M. (N=number of individuals). Statistical significance
was determined by one-way or two-way analysis of variance (ANOVA), followed by
post-hoc LSD analysis (Statistica 5.1; Statsoft, Tulsa, OK, USA).
Kinetic analysis of sodium influx was modelled using the MichaelisMenten equation, Jin=Jmax[Na+]/Km+[Na+], where Jmax is the maximal rate of sodium influx and Km is the sodium concentration at which sodium influx is half maximal. Values of Jmax and Km were taken directly from plots of sodium influx versus sodium concentration using SigmaPlot (ver. 8.0.2; SPSS, Inc.). Each curve represents the sodium influx of 56 individuals at each of 5 sodium concentrations.
Differences between kinetic parameters were determined by t-tests,
using the parameter and its S.E.M. as determined by best fit
MichaelisMenten analysis (Motulsky,
1998). A conservative approach was taken by treating each sodium
concentration, as opposed to each individual, as a single value. Consequently
each pairwise comparison was assessed with 8 degrees of freedom
[2(N1)]. This conservative approach compensated for the lack
of multiple comparison corrections, which were considered inappropriate due to
the inflated chance of type II error
(Perneger, 1998
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The kinetic parameters illustrated in Fig. 2 were derived from the curves shown in Fig. 1. The maximal rate of sodium influx (Jmax) was strongly influenced by calcium level and pH of the ambient water (Fig. 2A). In the absence of calcium, decreasing pH (increasing proton concentration) raised Jmax from a value of 1.2 ±0.09 µequiv mg1 wet mass h1 at pH 8 to 2.11±0.32 µequiv mg1 wet mass h1 at pH 6. The opposite effect was observed when calcium was high (1 mmol l1), with decreasing pH inhibiting maximal sodium influx. Low pH (4) also significantly reduced Jmax at intermediate calcium concentrations (0.5 mmol l1). Within pH treatments, calcium exerted significant actions on sodium influx capacity. At low pH elevated calcium levels inhibited sodium influx. At high pH increased calcium levels stimulated Jmax with a sodium influx of 1.20±0.09 µequiv mg1 wet mass h1 recorded for daphnids exposed to pH 8 and 0 mmol l1 calcium, compared to a value of 2.32±0.16 µequiv mg1 wet mass h1 for those exposed to pH 8 and 1 mmol l1 calcium in the ambient water.
|
Effects of calcium and pH on sodium influx affinity (Km) were less prevalent (Fig. 2B). Within calcium concentrations, pH had no influence on sodium uptake affinity. Comparisons between different calcium levels at a constant pH revealed an enhanced Km (decreased sodium influx affinity) with increased calcium levels at pH 6 and 8. A threefold increase in Km was observed at pH 6 when calcium was increased from 0 to 1 mmol l1 (69±47 vs. 207±49 µmol l1), while at pH 8, similar increases in calcium raised the Km tenfold (17±11 vs. 167±37 µmol l1).
The influence of calcium and pH on whole body sodium content reflected the effects observed on sodium influx (Fig. 3AC). At low pH, elevated sodium status was observed at low calcium levels. Conversely at low pH, significantly reduced whole body sodium contents were associated with high waterborne calcium levels. Whole body sodium concentrations at pH 4 and 1 mmol l1 calcium were in the order of 2025 mg kg1 wet mass, approximately 5075% of whole body sodium content at pH 8 and 1 mmol l1 calcium.
|
The effect of calcium on sodium influx was investigated further (Fig. 4). At both low (50 µmol l1) and high (1000 µmol l1) sodium levels there was no statistical difference between the two sodium salts tested. At low sodium levels (Fig. 4A), calcium inhibited sodium influx. Sodium influx was especially sensitive to inhibition by low levels of calcium. The addition of 100 µmol l1 calcium decreased sodium (as gluconate) influx from 0.82±0.21 to 0.49±0.08 µequiv g1 wet mass h1. Despite a 50-fold increase in calcium concentration, there was only minimal additional reduction in sodium influx.
|
At 1000 µmol l1 sodium (Fig. 4B), calcium levels up to 100 µmol l1 were again observed to inhibit sodium influx. This effect followed a similar pattern to that observed at 50 µmol l1 with a maximal inhibition of 54% noted at a calcium concentration of 100 µmol l1 for the sodium chloride experiment. This decrease was not, however, statistically significant. As calcium levels were raised further inhibition of sodium influx was not observed, and instead sodium influx rates were restored to control (calcium-free) levels.
Amiloride inhibited sodium influx at both low and high sodium
concentrations in a dose-dependent manner
(Fig. 5A). Maximal sodium
influx inhibitions of 93% (at 50 µmol l1 sodium) and 85%
(at 300 µmol l1 sodium) were reached at 5 mmol
l1 amiloride, with addition of higher amiloride
concentrations having no further effect on sodium influx. Pretreatment with
amiloride resulted in similar sodium inhibition effects (not shown). A Dixon
plot (Fig. 5B) was constructed
for amiloride concentrations up to 5 mmol l1 (maximal
inhibition). From this figure the inhibition constant (Ki)
for the effect of amiloride on sodium influx was calculated as 180 µmol
l1 amiloride. The amiloride concentration at which the two
lines intersect is the inverse of the Ki
(Cornish-Bowden, 1979).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the absence of calcium, pH had the opposite effect on sodium influx in Daphnia than that observed at 1 mmol l1 calcium. Under calcium-free conditions increasing proton concentration from pH 8 to 4 had no significant effect on sodium transport affinity (Km), yet stimulated sodium transport capacity (Jmax). This suggests the actions of protons at low calcium, in contrast to its actions at high calcium, are not associated with a competitive interaction between sodium and protons at the active transport site, and instead may be characteristic of a sodiumproton exchange mechanism.
In several invertebrate tissues the presence of an electrogenic
sodiumproton exchanger has been suggested by physiological
(Ahearn and Clay, 1989;
Shetlar and Towle, 1989
),
immunohistochemical (Kimura et al.,
1994
), molecular (Towle et
al., 1997
) and oocyte expression
(Mandal et al., 2001
) studies.
This apical exchanger, unlike that found in vertebrate and prokaryotic
systems, mediates the influx of two sodium ions in exchange for a single
proton (for a review, see Ahearn et al.,
2001
). It is further distinct from these exchangers in that it
exhibits strong calcium-dependence (Ahearn
and Franco, 1990
; Zhuang and
Ahearn, 1996
). This exchanger is capable of transporting calcium
across the apical surface and thus performs a potentially important role in
calcium homeostasis (Ahearn et al.,
2004
). It appears that calcium and sodium share the same transport
site, as these ions reciprocally inhibit the others passage in a competitive
manner (Ahearn and Franco,
1990
). Such a mechanism is consistent with the findings herein, of
reduced sodium influx at high calcium levels, due in part to a competitive
interaction (effect on Km).
Another important feature of this invertebrate electrogenic
2Na+/1H+ exchanger is its cooperativity. A distinctive
sigmoidal influx curve is generated as a function of increasing external
sodium levels (Ahearn and Clay,
1989; Shetlar and Towle,
1989
). In most invertebrate cell types and tissues where this
antiporter has been proposed to be responsible for sodium uptake, Hill
coefficients close to two are described (see
Ahearn et al., 2001
;
Mandal et al., 2003
),
suggesting cooperativity of sodium binding. In Daphnia sodium influx
was a hyperbolic, not a sigmoidal, function of external sodium concentration.
In the current study, sodium influx was monitored over a range of sodium
concentrations that are within a relevant range for a freshwater organism
(01500 µmol l1). This range is considerably lower
than that examined in previous investigations of uptake kinetics via
this exchanger (range from
2.5 up to 400 mmol l1).
Previous studies have focussed on sodium uptake in epithelia that are
routinely exposed to relatively sodium-enriched milieus (marine invertebrate
gills, gut; Ahearn et al.,
2001
). Sodium concentrations examined in Daphnia
therefore are at levels unlikely to generate sigmoidal uptake kinetics. Thus
the role of a putative 2Na+/H+ exchanger in facilitating
sodium influx in Daphnia cannot be excluded. This does, however,
suggest that in low sodium freshwaters the transporter may function according
to MichaelisMenten kinetics, transporting a single sodium ion across
the apical surface.
Cooperativity is generated by the existence of more than one sodium binding
site. This has been demonstrated in freshwater prawn hepatopancreas by
amiloride inhibition data showing two distinct binding sites via
Dixon plot analysis (Ahearn and Clay,
1989). While Shetlar and Towle
(1989
) described a single
inhibition constant of amiloride, suggesting a single sodium binding site,
they also noted that the square of the amiloride concentration gave a better
fit to the sodium influx inhibition data. Thus they suggested the presence of
two amiloride binding sites. Similarly, while a single Ki
of 180 µmol l1 was described for the effect of amiloride
on sodium influx in the present study, the square of the amiloride
concentration was a better fit (r2 0.9878 vs.
0.9924 for the low sodium treatment). Based on the interpretation of Shetlar
and Towle (1989
), the results
presented here could also be suggestive of a two-binding-site model, and again
could support the existence of a 2Na+/1H+ exchanger.
While sodium concentrations used in this experiment were likely insufficient to generate cooperativity, proton levels may have resulted in cooperative effects. The non-competitive stimulation of sodium transport observed in the absence of calcium and the presence of high proton concentration could be evidence of such an effect. Proton binding to one of the putative sodium binding sites may have acted to facilitate sodium binding to the other sodium binding site, thus promoting increased sodium influx, in a cooperative manner. In the presence of calcium (1 mmol l1) such a mechanism may not exist, due to the ability of calcium to block the sodium transport site.
Calcium may both inhibit and stimulate sodium influx
As discussed above, the competitive effects of calcium on sodium influx
appear to fit a mechanism of sodium influx that involves the invertebrate
electrogenic 2Na+/1H+ exchanger. The effect of calcium
on sodium influx in Daphnia magna over this relatively small range of
calcium levels (01 mmol l1) was extended to a larger
range of calcium concentrations (05 mmol l1). At a
low sodium concentration (50 µmol l1) the dose-dependent
inhibition of calcium was prominent, clear-cut and occurred at a relatively
low external calcium concentration (100 µmol l1). At a
higher sodium concentration (1000 µmol l1), inhibition
was also discerned up until a calcium level of 100 µmol
l1. Thereafter, however, increasing calcium stimulated
sodium influx. This suggests the possibility of a calcium-stimulated sodium
uptake pathway that initially negates, then supersedes the inhibitory actions
of calcium at lower sodium and calcium levels. The presence of apical
sodium/calcium exchange has been described in invertebrate tissues
(Zhuang and Ahearn, 1996).
This transporter would likely only be active when calcium levels are high, and
may serve as a mechanism for regulating intracellular calcium. As calcium is
transported out of the cell, sodium would move into the cell, thus influx
stimulation would be observed. As this mechanism only appears to operate at
relatively high sodium levels it suggests this pathway may have a
comparatively low affinity for sodium, and thus may be of limited
physiological relevance as a route of sodium influx in Daphnia.
The results of the calcium inhibition study also showed that there was no
effect of anion on sodium influx. Sodium influx data were statistically
identical when either chloride or gluconate sodium salts were used. In fish
and other invertebrates sodium and chloride transport is independent, although
often linked (Towle, 1993;
Evans et al., 1999
). The data
presented here support the chloride-independence of sodium influx in
Daphnia magna.
Comparison with other aquatic organisms
Disturbances in sodium balance with pH have been well documented in both
fish and decapod crustaceans (see
Vangenechten et al., 1989;
Wood, 1989
). By contrast,
little is known regarding the effect of pH on sodium metabolism in smaller
freshwater crustaceans. Potts and Fryer
(1979
) described inhibited
sodium uptake with low pH in two Cladoceran species, while Havas et al.
(1984
) noted inhibitory
effects of acid on sodium metabolism in daphnids exposed to soft water. These
latter authors also demonstrated that the sodium influx component of sodium
metabolism exhibited greater inhibition in soft than in hard water
(Havas et al., 1984
), a
pattern somewhat contrary to that observed in the present study. The
conclusions in this study were somewhat confounded by testing the effect of
calcium in natural waters that varied considerably in sodium content
(Havas et al., 1984
). As
sodium uptake is a saturable, facilitated process, the response of the sodium
concentration tested will be highly dependent on its relationship to the
affinity and capacity of the transport process, stressing the value of the
kinetic approach used herein.
It was, nevertheless, somewhat surprising in our study that high calcium,
and low pH, eliminated sodium influx in Daphnia magna. It has been
well known for some time that calcium is an important ameliorator of the
physiological perturbations induced by exposure of freshwater fish to acid
waters (see Wood, 1989). In
laboratory experiments the protective role of increased calcium levels has
been explained in terms of a restoration of branchial tight junction
integrity, compensating for the initial displacement of junctional calcium by
high proton levels (McDonald et al.,
1980
; McDonald,
1983
). The increased calcium thus acts to limit the enhanced
sodium efflux component caused by acid water. In freshwater crustaceans,
however, it appears that the influx, not the efflux, component is the primary
mediator of lowered sodium status. Whole body sodium levels in the current
study mirrored trends in sodium influx closely, suggesting little influence of
sodium efflux (Fig. 1 cf.
Fig. 3). In the freshwater
crayfish Orconectes, it was noted that sodium efflux was relatively
acid-resistant (Wood and Rogano,
1986
), supporting the results of an earlier study
(Shaw, 1960
). Furthermore,
Potts and Fryer (1979
) failed
to delineate any significant effect of calcium on acid-induced changes in
sodium efflux in Daphnia magna and Acantholeberis
curvirostris. It is also of interest to note that increased water
hardness appears to offer little protective effect against the toxicity of the
sodium antagonist, silver, to Daphnia magna (e.g.
Bury et al., 2002
). Evidence
therefore suggests that freshwater crustaceans are fundamentally different
from freshwater fish in terms of their physiological response to the modifying
influence of calcium on acid-exposed sodium metabolism.
In direct contrast to fish, it is only in hard water that acidification of
the medium becomes problematic with regards to sodium influx in Daphnia
magna. This could be related to calcium metabolism. Daphnia
magna moult every 24 days
(Peltier and Weber, 1993).
Each moulting event is associated with massive fluxes in mineral status, as
the exoskeleton is sloughed and a new exoskeleton is remineralised
(Wheatly and Gannon, 1995
).
Given the relatively low external calcium levels associated with freshwaters
and the high frequency of Daphnia moulting, it is likely that
considerable ion uptake resources are devoted to effective calcium scavenging.
The severe inhibition of sodium uptake in the presence of high calcium
conditions, as possibly mediated by competitive interactions at a
2Na+/H+ exchanger, could explain why Daphnia
differ from freshwater fish, and indeed other freshwater crustaceans with
longer moulting cycles. Ellis and Morris
(1995
) ascribed an apparently
anomalous ion regulation response to acidification in a freshwater crayfish to
perturbation in calcium metabolism, supporting a similar conclusion in
Daphnia.
Methodological considerations
There are considerable methodological difficulties associated with studying
sodium transport mechanisms in Daphnia magna. Their small size limits
the ability to examine sodium influx in isolated tissues, cell types or
membrane surfaces. All of these techniques have been crucial for an
understanding of sodium metabolism in fish and euryhaline crustaceans (see
Evans et al., 1999;
Ahearn et al., 2001
).
In the current study sodium influx was examined across the whole animal. Sodium influx thus represents the summed effects of sodium taken up across the epipodite apical surface, that transported across the basolateral surface to the haemolymph, and also sodium that may be absorbed via the gastrointestinal pathway. Each of these membrane barriers to transport may handle sodium by distinct mechanisms, resulting in whole body sodium uptake patterns that may differ somewhat from sodium uptake discerned in larger organisms using homogenous preparations. Nevertheless the data presented here suggest that sodium influx in Daphnia magna is achieved by sodiumproton exchange, a mechanism that would explain the sensitivity of these organisms to acidified environments and waterborne calcium levels.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahearn, G. A. and Clay, L. P. (1989). Kinetic analysis of electrogenic 2Na+-1H+ antiport in crustacean hepatopancreas. Am. J. Physiol. 257,R484 -R493.[Medline]
Ahearn, G. A. and Franco, P. (1990). Sodium and calcium share the electrogenic 2Na+-1H+ antiporter in crustacean antennal gland. Am. J. Physiol. 259,F758 -F767.[Medline]
Ahearn, G. A., Mandal, P. K. and Mandal, A. (2001). Biology of the 2Na+/1H+ antiporter in invertebrates. J. Exp. Zool. 289,232 -244.[CrossRef][Medline]
Ahearn, G. A., Mandal, P. K. and Mandal, A. (2004). Calcium regulation in crustaceans during the molt cycle: a review and update. Comp. Biochem. Physiol. 137A,247 -257.
Bianchini, A. and Wood, C. M. (2003). Mechanisms of acute silver toxicity in Daphnia magna. Environ. Toxicol. Chem. 22,1361 -1367.[CrossRef][Medline]
Bury, N. R., Shaw, J., Glover, C. and Hogstrand, C. (2002). Derivation of a toxicity-based model to predict how water chemistry influences silver toxicity to invertebrates. Comp. Biochem. Physiol. 133C,259 -270.
Cornish-Bowden, A. C. (1979). Fundamentals of Enzyme Kinetics. London: Butterworth and Co.
Ellis, B. A. and Morris, S. (1995). Effects of extreme pH on the physiology of the Australian yabby Cherax destructor: acute and chronic changes in haemolymph carbon dioxide, acidbase and ionic status. J. Exp. Biol. 198,395 -407.[Medline]
Evans, D. H., Piermarini, P. M. and Potts, W. T. W. (1999). Ionic transport in fish gill epithelium. J. Exp. Zool. 283,641 -652.[CrossRef]
Glover, C. N., Pane, E. F. and Wood, C. M. (2005). Humic substances influence sodium metabolism in the freshwater crustacean, Daphnia magna. Physiol. Biochem. Zool., in press.
Havas, M., Hutchinson, T. C. and Likens, G. E. (1984). Effect of low pH on sodium regulation in two species of Daphnia. Can. J. Zool. 62,1965 -1970.
Kimura, C., Ahearn, G. A., Busquets-Turner, L., Haley, S. R.,
Nagao, C. and de Couet, H. G. (1994). Immunolocalisation of
an antigen associated with the invertebrate electrogenic
2Na+/1H+ antiporter. J. Exp.
Biol. 189,85
-104.
Kirschner, L. B. (2004). The mechanism of
sodium chloride uptake in hyperregulating aquatic animals. J. Exp.
Biol. 207,1439
-1452.
Leivestad, H., Hendrey, G., Muniz, I. P. and Snekvik, E. (1976). Effects of acid precipitation on freshwater organisms. In Impact of Acid Precipitation on Forest and Freshwater Ecosystems in Norway (ed. F. H. Braekke). Oslo: SNSF Project FR6/76.
Lin, H. and Randall, D. J. (1995). Proton pumps in fish gills. In Fish Physiology. XIV. Cellular and Molecular Approaches to Fish Ionic Regulation (ed. C. M. Wood and T. J. Shuttleworth), pp. 229-255. New York: Academic Press.
Mandal, A., Mandal, P. and Ahearn, G. A. (2001). Transport of 22Na+ and 45Ca2+ by Xenopus laevis oocytes expressing mRNA from lobster hepatopancreas. J. Exp. Zool. 290,347 -358.[CrossRef][Medline]
Mandal, P. K., Mandal, A. and Ahearn, G. A. (2003). Differential physiological expression of the invertebrate 2Na+/1H+ antiporter in single epithelial cell type suspensions of lobster hepatopancreas. J. Exp. Zool. 297A,32 -44.[CrossRef]
McDonald, D. G. (1983). The interaction of environmental calcium and low pH on the physiology of the rainbow trout, Salmo gairdneri. J. Exp. Biol. 102,123 -140.
McDonald, D. G., Hbe, H. and Wood, C. M.
(1980). The influence of calcium on the physiological responses
of the rainbow trout, Salmo gairdneri, to low environmental pH.
J. Exp. Biol. 88,109
-131.[Abstract]
Motulsky, H. (1998). Comparing dose-response or kinetic curves with GraphPad Prism. HMS Beagle 34, (http://hmsbeagle/34/booksoft/softsol.htm).
Peltier, W. and Weber, C. I. (1993).Methods for Measuring the Acute Toxicity of Effluents to Freshwater and Marine Organisms, 4th edn. Cincinnati: US Environmental Protection Agency, EPA 600/4-90/027F.
Perneger, T. V. (1998). What's wrong with
Bonferroni adjustments. Br. Med. J.
316,1236
-1238.
Potts, W. T. W. and Fryer, G. (1979). The effects of pH and salt content on sodium balance in Daphnia magna and Acantholeberis curvirostris (Crustacea: Cladocera). J. Comp. Physiol. 129,289 -294.
Potts, W. T. W. and Parry, G. (1964). Osmotic and Ionic Regulation in Animals. Oxford: Pergamon Press.
Shaw, J. (1960). The absorption of sodium ions by the crayfish Astacus pallipes Lereboullet. III. The effect of other cations in the external solution. J. Exp. Biol. 37,548 -556.
Shetlar, R. E. and Towle, D. W. (1989). Electrogenic sodium-proton exchange in membrane vesicles from crab (Carcinus maenas) gill. Am. J. Physiol. 257,R924 -R931.[Medline]
Stobbart, R. H., Keating, J. and Earl, R. (1977). A study of sodium uptake by the water flea Daphnia magna. Comp. Biochem. Physiol. 58A,299 -309.
Towle, D. W. (1993). Ion transport systems in membrane vesicles isolated from crustacean tissues. J. Exp. Zool. 265,387 -396.
Towle, D. W., Rushton, M. E., Heidysch, D., Magnani, J. J.,
Rose, M. J., Amstutz, A., Jordan, M. K., Shearer, D. W. and Wu, W.-S.
(1997). Sodium/proton antiporter in the euryhaline crab
Carcinus maenas: molecular cloning, expression and tissue
distribution. J. Exp. Biol.
200,1003
-1014.
Vangenechten, J. H. D., Witters, H. and Vanderborght, O. L. J. (1989). Laboratory studies on invertebrate survival and physiology in acid waters. In Acid Toxicity and Aquatic Animals (ed. R. Morris, E. W. Taylor, D. J. A. Brown and J. A. Brown), pp. 153-169. Cambridge: Cambridge University Press.
Wheatly, M. G. and Gannon, A. T. (1995). Ion regulation in crayfish: freshwater adaptations and the problem of molting. Am. Zool. 35,49 -59.
Wood, C. M. (1989). The physiological problems of fish in acid waters. In Acid Toxicity and Aquatic Animals (ed. R. Morris, E. W. Taylor, D. J. A. Brown and J. A. Brown), pp. 125-152. Cambridge: Cambridge University Press.
Wood, C. M., La Point, T. W., Armstrong, D. E., Birge, W. J., Brauner, C. J., Brix, K. V., Call, D. J., Crecelius, E. A., Davies, P. H., Gorsuch, J. W., Hogstrand, C., Mahony, J. D., McGeer, J. C. and O'Connor, T. P. (2002). Biological effects of silver. In Silver in the Environment: Transport, Fate, and Effects (ed. A. W. Andren and T. W. Bober). Pensacola: Society of Environmental Toxicology and Chemistry Press.
Wood, C. M. and Rogano, M. S. (1986). Physiological response to acid stress in crayfish (Orconectes): haemolymph ions, acid-base status, and exchanges with the environment. Can. J. Fish. Aquat. Sci. 43,1017 -1026.
Zhuang, Z. and Ahearn, G. A. (1996).
Ca2+ transport processes of lobster hepatopancreatic brush-border
membrane vesicles. J. Exp. Biol.
199,1195
-1208.