Water calcium concentration modifies whole-body calcium uptake in sea bream larvae during short-term adaptation to altered salinities
1 Centre of Marine Sciences (CCMAR), University of Algarve, Campus de
Gambelas, 8005-139 Faro, Portugal
2 Department of Animal Physiology, University of Nijmegen, 6525 ED Nijmegen,
The Netherlands
* Author for correspondence (e-mail: acanario{at}ualg.pt)
Accepted 22 October 2003
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Summary |
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Key words: calcium uptake, drinking rate, salinity, environmental calcium, larvae, gilthead sea bream, Sparus auratus
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Introduction |
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Internal [Ca2+] is maintained within narrow limits despite large
fluctuations in environmental [Ca2+] that generally parallel
changes in water salinity. The ability to maintain a constant ion
concentration and osmolality of body fluids appears early in development.
Embryos and larvae have a capacity similar to that of adult fish and regulate
ionic gradients between their body and the ambient water by excreting sodium
chloride or absorbing calcium (Alderdice,
1988; Guggino,
1980
). The early post-embryonic stages of the European sea bass
(Dicentrarchus labrax) are able to hypo- and hyper-regulate over a
wide range of salinities, this ability being acquired in steps but already
being present at hatching (Varsamos et
al., 2001
). It has also been proposed that the regulation of
calcium balance in freshwater tilapia larvae (Oreochromis
mossambicus) acclimated to low-calcium environments is stage dependent
and that modulation of calcium fluxes is closely correlated with levels of
body calcium content (Chou et al.,
2002
; Hwang et al.,
1996
).
Fish in seawater tend to gain ions such as sodium and chloride through
diffusion and to lose water by osmosis. The acquisition of the capacity to
control water balance in relation to external salinity through drinking by
fish larvae is, therefore, fundamental for osmoregulation
(Flik et al., 2002;
Varsamos et al., 2001
), and
drinking occurs before the development of a functional anus and gills (Tytler
and Blaxter,
1988a
,b
).
Since whole-body calcium uptake correlates positively to environmental [Ca2+], salinity (as a result of [NaCl]) and drinking, the relative contribution of each of these variables to calcium uptake is uncertain. In the present study, we have examined the relationship between calcium uptake and drinking by submitting gilthead sea bream larvae to abrupt short-term changes of combinations of water salinity and [Ca2+].
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Materials and methods |
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Determination of calcium influx
Fish were netted and transferred to 25-ml vessels with aerated seawater
and, after 30 min, 45CaCl2 (NEN, Boston, MA, USA) was
added to the water (final activity of 3.7 kBq ml1). At the
end of the experiment (normally lasting 4 h), water samples were collected.
The larvae were rinsed in tracer-free water, sacrificed, weighed and digested
as indicated above, neutralised with an identical volume of 2 mol
l1 sodium hydroxide (Sigma-Aldrich) and bleached with 300
µl of 35% hydrogen peroxide (Fluka, Sigma-Aldrich) to prevent colour
quenching. Water calcium content was measured by a colourimetric endpoint
assay (Sigma-Aldrich, assay No. 587). Water and digested larval samples were
dissolved in OptiPhase HiSafe II liquid scintillation fluid (Wallac, Amersham
Pharmacia Biotech, Lisbon, Portugal), and 45Ca activity was
measured in a scintillation counter (Model LS6000IC; Beckman Instruments Inc.,
Fullerton, CA, USA). Calcium influx (Cain) was calculated according
to the following equation: Cain=
(AfCw)/(Awt),
where Af is the total 45Ca activity in fish
(c.p.m.), Cw is the total calcium concentration in water
(nmol l1), Aw is the total
45Ca activity in water (c.p.m. l1), and
t is duration of exposure (h). Calcium influx is expressed as nmol
h1. Extra-intestinal calcium uptake was estimated by
subtracting the amount of calcium in the water imbibed (i.e. drinking rate;
see below) from the total whole-body calcium uptake.
Determination of drinking rate
The procedure used for the determination of drinking rate was the same as
for calcium influx but 3.7 kBq ml1 of 51Cr-EDTA
(NEN) was added to the water. Radioactivity was counted in a gamma counter
(Wallac 1470 Wizard gamma counter; Amersham Pharmacia Biotech). In some
experiments, calcium influx and drinking rate were measured simultaneously.
Drinking rate (DR) was calculated as:
DR=Af/(Awt), where
Af is the total activity of 51Cr-EDTA in the
fish (c.p.m.), Aw is the tracer activity in water (c.p.m.
ml1), and t is the duration of exposure (h).
Results are expressed as nl h1.
Effects of tracer exposure time and body size on whole-body Ca2+ influx
The effect of duration of exposure to 45CaCl2 on
calcium uptake was determined by sampling larvae in different vessels at 2 h,
4 h, 6 h, 8 h and 16 h after radioisotope addition. Intact gilthead sea bream
larvae accumulated 45Ca at a constant rate for at least 16 h and
therefore the tracer experiments were conducted within this time-span to
reflect initial speed of calcium influx, thus eliminating possible effects of
tracer backflow.
The effect of body size was tested in five different experiments in 100%SW with larvae ranging in mass from 5 mg to 150 mg. Calcium influx was determined as described above.
Time-series of adjustment of Ca2+ influx to diluted seawater
Larvae were transferred to 25-ml vessels containing 100%SW (four groups,
N15) or 50%SW (three groups, N
15), and
45CaCl2 was immediately added. Larvae were collected at
2 h, 4 h, 8 h and 16 h after transfer (HAT; 100%SW) and at 4 HAT, 8 HAT and 16
HAT (50%SW), rinsed, sacrificed, weighed and dissolved to determine calcium
influx as described above.
Effects of [Ca2+] and [Na+] on Ca2+ influx and drinking rates
Gilthead sea bream larvae (N=2040 per group) were
transferred to 25-ml vessels containing the experimental media (see
Table 1 for precise ionic
composition): 100%SW, 150%SW, 25%SW, 10%SW, 0%SW, 100%SW plus CaCl2
(100%SW+Ca), 25%SW plus CaCl2 (25%SW+Ca), 25%SW plus NaCl
(25%SW+Na), 10%SW plus CaCl2 (10%SW+Ca) and 10%SW plus NaCl
(10%SW+Na) for 4 h and 20 h (0%SW only for 4 h) before addition of
45CaCl2 and 51Cr-EDTA. After 4 h in
radioisotope-containing water, the fish were rinsed, sacrificed, weighed and
dissolved to determine calcium influx and drinking rate. [Ca2+] in
100%SW+Ca was adjusted to that of 150%SW, and 25%SW+Ca and 10%SW+Ca was
adjusted to that of 100%SW. [Na+] in 25%SW+Na and 10%SW+Na was
adjusted to that of 100%SW.
Statistical analysis
The effect of treatments on calcium uptake and drinking was analysed by
one-way or two-way covariance analysis (ANCOVA) with body size as covariate
using the General Linear Model module in the Systat software (version 10.2;
Systat Software Inc., Richmond, CA, USA). Contrasts were used to test the
relationship among cell means. Least squares means and their standard errors
adjusted for mass are used to report effect of treatments. Multiple linear
regression analysis was used to compare the relative contribution of calcium,
sodium and osmolarity to calcium uptake. Whenever the data were required to be
normalised, a logarithmic transformation was used. The significance level was
established as P<0.05 unless otherwise stated.
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Results |
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Effects of salinity and addition of Ca2+ or Na+ on Ca2+ influx
Whole-body calcium influx in 100%SW was strongly dependent on body mass
(Cain=2588M0.496;
Fig. 1A), even after
normalisation to unit of body mass
(Cain,M=2588M504;
Fig. 1B). The same relationship
was observed 8 HAT to media of other salinities; the slope was similar but the
y-intercept was proportional to the salinity (data not shown).
Because the dependency of calcium influx on body mass was highly significant
across the mass range of the larvae used in the experiments, subsequent data
analysis considered mass as a covariate to remove its effect. When fish were
transferred from 100%SW to 50%SW, adjustment to a lower calcium influx was
noticeable between 4 h and 8 h after the onset of exposure
(Fig. 2). By 8 HAT, the influx
rate at 50%SW was 30% significantly lower than at 100%SW and was 65% lower by
16 HAT. Transfer of larvae from 100%SW to 150%SW led to a 50% increase in
calcium influx by 8 HAT (Fig.
3A) and 100% by 24 HAT (Fig.
3B). Transfer to lower salinities lowered calcium influx 8 HAT by
50% at 25%SW and by 90% at 0%SW (Fig.
3A). A similar reduction was also found 24 HAT.
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To establish whether salinity or ambient [Ca2+] determined the rate of calcium uptake, larvae were exposed to altered salinities and to different [Ca2+] and [Na+]. Larvae exposed to 100%SW to which [Ca2+] was adjusted to 17.6 mmol l1 (similar to that of 150%SW) had calcium influx rates at 8 HAT and 24 HAT similar to those in 150%SW (Fig. 3A,B).Similarly, transfer to 25% and 10% diluted seawater in which [Ca2+] had been adjusted to levels of 100%SW resulted in increased calcium influx rates. At 8 HAT, the increase was only significant at 10%SW+Ca (although influx rates did not reach those of 100%SW), but at 24 HAT both 25%SW+Ca and 10%SW+Ca had calcium influx rates similar to those found in 100%SW (Fig. 3B).
Placing larvae in diluted medium supplemented with NaCl to levels similar to 100%SW resulted in high mortality (Table 2), which was most severe in the 10%SW+Na group: all fish died within 2 HAT. Nevertheless, the surviving 25%SW+Na group had calcium influx rates similar to their respective controls (25%SW) at both 8 HAT and 24 HAT.Interestingly, fish transferred directly to 0%SW were able to adjust better than fish in 10%SW+Na, with 50% of the initial group surviving to the end of the 8-h period. To confirm that calcium influx was related only to fish size and to water [Ca2+], a multiple regression analysis, which also included water osmolarity and [Na+] as independent variables, was carried out. We found strong colinearity between medium [Ca2+], [Na+] and osmolarity, implying some uncertainty as to the relative contributions between the three variables to calcium influx. The multiple linear regression equation, pooling data from 8 HAT and 24 HAT, was: log Cain=2.659+0.003[Na+]0.001Os+0.041[Ca2+]+0.679 log M, where Os is osmolarity. As can be observed, according to this model, the effect of [Ca2+] on Ca2+ influx is at least one order of magnitude higher than that of [Na+] or osmolarity, suggesting that ambient [Ca2+] is a major modifier of Ca2+ influx. The relationships between calcium influx, body mass and medium [Ca2+] at 8 HAT and 24 HAT were as follows: Cain,8=283+M0.748+[Ca2+]0.618 (adjusted r2=0.511, N=213, P<0.001; range [Ca2+] 0.517.6 mmol l1; range M 8.682.8 mg) and Cain,24=813+ M0.575+[Ca2+]0.424 (adjusted r2=0.511, N=128, P<0.001; range [Ca2+] 1.6817.6 mmol l1; range M 10.057.6 mg).
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Effect of salinity and addition of [Ca2+] or [Na+] on drinking
As with calcium uptake, drinking rate was highly positively correlated to
fish mass (at 100%SW DR=46.6M0.785;
r0=0.677, P<0.001, N=78). A marked
salinity-dependent reduction in drinking was observed at 8 HAT
(Fig. 4A). Fish that were
exposed to media at lower salinities exhibited drinking rates that were
significantly lower than those of control 100%SW fish. Fish exposed to 150%SW
had drinking rates that were slightly higher but not statistically different
from those kept at 100%SW. Drinking at 0%SW was significantly reduced in
relation to all other tested media. After 24 HAT, similar relationships to
that of 8 HAT were observed (Fig.
4B).Addition of calcium or sodium had no significant effects on
drinking, although there was an indication that addition of calcium and
especially sodium might stimulate drinking after 24 h. However, the large
variability and low number of surviving fish meant this trend could not be
confirmed.
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Calcium imbibed was estimated from the drinking rate, and the
extra-intestinal contribution to the whole-calcium uptake was determined
(Fig. 5). Extra-intestinal
contributions were higher in media with lower salinity and less calcium,
accounting for 93% of whole-body calcium entry at 10%SW and 0%SW and for
85% at 25%SW. At 100%SW and above, the extra-intestinal contribution was
only 4550%. The effect of adding calcium to the water at lower
salinities was to increase the relative contributions to levels close to those
of 100%SW. However, addition of Na had little effect. The change in the
relative contribution of the two routes of calcium uptake is summarized in
Fig. 6. The data further
emphasise that increased calcium uptake at higher salinities is intimately
associated with drinking but it is also dependent on environmental
[Ca2+], since extra-intestinal uptake is also enhanced. It was not
possible to separate the relative importance of drinking and [Ca2+]
because of colinearity in the multiple regression.
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Discussion |
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Since extracellular calcium is maintained within tight limits, this
indicates that gilthead sea bream larvae are able to regulate their calcium
uptake (and loss) in order to comply with the calcium demands of their normal
physiological processes as well as the extra demand imposed by the intense
calcification period. This is also supported by the observation that fish
adapted to low-calcium environments are capable of modulating transport
mechanisms to sustain similar or greater accumulation rates of body calcium
content during growth (Chou et al.,
2002; Flik et al.,
1986
; Hwang et al.,
1996
; Mol et al.,
1999
; Vonck et al.,
1998
).
Gilthead sea bream larvae transferred from 100%SW to a hyposmotic
environment (50%SW) require at least 16 h to adapt calcium transport
mechanisms to the new environmental conditions. In juveniles of silver sea
bream (Sparus sarba), abrupt hyposmotic exposure (from 33 to
6
) resulted in a decline in serum total calcium at 24 HAT and a return
to pre-exposure levels by 120 HAT (Kelly
and Woo, 1999
). A similar effect was reported by Mancera et al.
(1993
) for the gilthead sea
bream, with complete recovery occurring after 30 days. In gilthead sea bream
larvae, it was not possible to measure serum calcium, but a strong
relationship between calcium influx and water salinity was found both at 8 HAT
and 24 HAT. Furthermore, [Ca2+] appears to be the main
environmental factor determining calcium influx, as shown by the fact that
fish exposed to CaCl2-enriched seawater had whole-body influx rates
similar to those of control (non-enriched) seawater of similar calcium
content. By contrast, salinity itself appeared to have little effect on
calcium influx since NaCl-enriched diluted seawater had no effect on calcium
influx. However, an effect of NaCl on calcium influx cannot be completely
excluded since the low number of surviving larvae in the NaCl-treated group
may not have allowed a more thorough analysis.
Few studies have described the effects of environmental calcium on calcium
uptake in fish larvae. Tilapia larvae acclimated to freshwater with a high or
low calcium content had a differential calcium influx when exposed to diverse
calcium concentrations (Hwang et al.,
1994). Tilapia larvae, in common with the sea bream larvae, had
significantly lower calcium influxes immediately after transfer to water
containing less calcium. Moreover, effluxes became progressively lower in the
low-calcium group, indicating calcium retention to maintain a positive balance
(Chou et al., 2002
;
Hwang et al., 1996
). These
observations further emphasise the ability of larval fish to accommodate their
transporting mechanisms in response to changes in calcium.
An unexpected observation in the present study was that NaCl supplementation of diluted SW to obtain 36 g l1 resulted in very high mortalities. This suggests that ion-exchange mechanisms may have been affected and that the ratio between Na+ and/or Cl and the other ionic components in the water are essential for maintenance of normal physiology in larvae. Potential explanations for the effects observed with excess Na+ could be (1) a Na+/K+ imbalance that would compromise the Na+/K+ pump or (2) slowing down or even reversal in direction of the Na+/Ca2+ exchanger in the serosa side leading to an accumulation of intracellular Na+, which would be aggravated by the mentioned K+ imbalance. However, further work will be required to clarify which mechanisms are affected by NaCl supplementation.
Drinking and calcium uptake
The results from the present study with gilthead sea bream larvae show a
clear dependence of drinking rates on external osmolarity during the
adaptation of gilthead sea bream to low-salinity environments. This is
expected since the dehydrating strength of the medium determines the amount of
water required to replace osmotic losses through body surfaces
(Fuentes and Eddy, 1997).
Other studies with marine and euryhaline fish larvae herring
(Clupea harengus), plaice (Pleuronectes platessa), cod
(Gadus morhua; Tytler and
Blaxter, 1988a
), sea bass (Flik
et al., 2002
) and tilapia (Lin
et al., 2001
; Miyazaki et al.,
1998
) have similarly shown a decrease or increase in
drinking when exposed to lower or higher salinities, respectively. In the sea
bream larvae, exposure to 150%SW did not increase drinking rates significantly
above those of fish in 100%SW, possibly because salt loads would be too high,
as indicated by the higher mortality at 150%SW. Reduction in drinking at
55
, compared with 34
, has been observed in adult Aphanius
dispar in the Dead Sea, probably as a means to reduce salt intake
(Skadhaug and Lotan,
1974
).
In seawater fish, the amount of calcium that enters the intestinal tract as
a consequence of drinking is rather large, both due to high drinking rates and
the elevated calcium concentration in water
(Flik et al., 1995;
Flik and Verbost, 1993
;
Sundell and Björnsson,
1988
). There are no available data on calcium absorption from the
intestine in larvae and only a few studies have considered this parameter in
adult fish. Estimates of the contribution of intestinal calcium uptake are
variable and range from almost zero to 20% in freshwater and seawater tilapia
(Schoenmakers et al., 1993
) to
40% in seawater cod (Sundell and
Björnsson, 1988
) and 70% in the euryhaline flounder
Paralichthys lethostigma
(Hickman, 1968
). However, it
is likely that intestinal Ca2+ absorption rates vary with ambient
salinity, Ca2+ or bicarbonate
(Wilson et al., 2002
)
concentrations. Estimates in gilthead sea bream juveniles in 100%SW indicate
that the intestinal calcium absorption rate can reach 90%
(Guerreiro et al., 2002
).
Assuming an identical absorption rate in gilthead sea bream larvae, intestinal
calcium transport could account for as much as 40% of the total calcium
uptake. In the gilthead sea bream, the dependence of whole-body calcium uptake
and environmental [Ca2+] is mainly due to a high dependence of the
intestinal route on environmental conditions. The decrease in the intestinal
contribution with the decrease in salinity most probably was a result of a
limitation imposed by the amount of drinking, which can be enhanced or reduced
in proportion to environmental [Ca2+]. At lower environmental
[Ca2+], calcium influx is largely dependent on extra-intestinal
transport. These results are in agreement with those obtained by Vonck et al.
(1998
) for branchial calcium
influxes in juvenile tilapia raised at different salinities and water calcium
content. The lack of parallelism in the relationship between extra-intestinal
and intestinal calcium uptake as a function of [Ca2+] and the
smaller slope for extra-intestinal uptake
(Fig. 6) suggest that the
dominant uptake transport mechanisms at the two sites are different, perhaps
with active transport being an important component extra-intestinally and more
passive or exchange mechanisms favoured intestinally when calcium gradients
are favourable. In support for this hypothesis, a regulated low-affinity,
high-capacity transport, possibly mediated by a carrier protein, have been
described (Klaren et al.,
1993
). The question as to whether intestinal transport is just a
consequence of drinking or provides a significant contribution to calcium
balance remains to be clarified.
In fish larvae, the mitochondria-rich chloride cells, the major site for
Ca2+ and NaCl exchange in the external epithelia (Flik et al.,
1995,
1996
;
Perry and Flik, 1988
), are
located throughout the body integument, namely in areas where the interface
between the bathing water and the extracellular fluids is thin.
Seawater-adapted larvae have more chloride cells than those in freshwater,
presumably to excrete excess NaCl (Flik et
al., 2002
; van der Heijden et
al., 1999
). As fish larvae develop, the distribution of the
chloride cells changes and they become restricted to the gills in juveniles
(see review by Kaneko et al.,
2002
; van der Heijden et al.,
1999
; Wales and Tytler,
1996
). In 30-day-old gilthead sea bream larvae adapted to
seawater, the highest density of chloride cells (MR-cells) is present in
regions with high calcium requirements such as the jaw and fin epithelia,
which are undergoing mineralisation (P.M.G., J.F., G.F., J.R., D.M.P. and
A.V.M.C., unpublished results). The identification of extra-branchial MR-cells
in these regions, as well as in the trunk and opercular surface, suggests that
in gilthead sea bream larvae they may play an important role in
extra-intestinal calcium uptake, providing for direct uptake in areas of high
calcium demand, as previously suggested
(Flik et al., 1995
;
Hwang et al., 1994
;
Varsamos et al., 2002
).
In conclusion, the present study shows that gilthead sea bream larvae are able to accommodate their calcium-transporting mechanism according to the environmental conditions and that drinking and the intestine provide the main route for the increased calcium influx at higher salinities. The contribution of the intestine to the overall calcium uptake in diluted seawater and freshwater is negligible, and extra-intestinal mechanisms ensure adequate calcium uptake in calcium-depleted environments. By contrast, in calcium-rich waters and in seawater, the intestinal route becomes increasingly important. The mechanism by which calcium is taken up in the intestine remains to be elucidated but the strong uptake in response to added calcium and the relationship with salinity suggests that calcium transport is associated with or dependent on that of other ions.
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Acknowledgments |
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