Calcium handling in Sparus auratus: effects of water and dietary calcium levels on mineral composition, cortisol and PTHrP levels
1 Department of Animal Physiology, Faculty of Science, Radboud University
Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
2 Centre of Marine Sciences (CCMAR), University of Algarve, Campus de
Gambelas, 8005-139 Faro, Portugal
* Author for correspondence (e-mail: gertflik{at}sci.kun.nl)
Accepted 17 August 2004
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Summary |
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Key words: calcium balance, PTHrP, cortisol, hypocalcemia, growth, phosphorus, Sparus auratus
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Introduction |
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In fish blood, calcium is either complexed (e.g. to citrate), protein bound
or present as free ion. The free calcium fraction accounts for about half of
the total calcium fraction and is the physiologically important fraction
(Hanssen et al., 1991). Fish
regulate their ionic plasma calcium level more strictly than their
protein-bound calcium level, and this may relate to the fact that even minor
disruptions in ionic calcium concentrations lead to severe stress and
disturbance of calcium balance (Flik et
al., 1995
).
Unlike terrestrial vertebrates, which depend solely on the diet as their
calcium source, fish live in an environment with a readily available source of
calcium. Seawater has a calcium concentration of 10 mmol l-1,
whereas the total plasma calcium concentration of marine fish ranges from 2 to
3 mmol l-1; thus, marine fish live in a hypercalcic environment and
face an inward gradient of Ca2+. As calcium availability in the
environment varies, fish have developed calcium regulatory systems that can
react rapidly to changes in environmental calcium concentrations
(Wendelaar Bonga and Pang,
1991
; Bjornsson et al.,
1999
).
Endocrine control of calcium metabolism in fish is regulated by both hyper-
and hypocalcemic hormones. Stanniocalcin
(Lafeber et al., 1988;
Wagner et al., 1998
) acts as
the major hypocalcemic (in fact anti-hypercalcemic, as it inhibits
Ca2+ influx) hormone. Increased calcium levels in the medium induce
hypercalcemic conditions and, by doing so, promote stanniocalcin release into
the bloodstream, where it reduces the calcium influx in the gills and
intestine. Prolactin (Kaneko and Hirano,
1993
; Mancera et al.,
1993
; Flik et al.,
1994
) and PTHrP (parathyroid hormone related protein;
Guerreiro et al., 2001
) act as
major hypercalcemic hormones. PTHrP is phylogenetically the predecessor of
PTH, which appeared only after the water/land transition of vertebrates.
Although recent reports indicate that fish express PTH
(Danks et al., 2003
;
Gensure et al., 2004
), they
also have PTHrP, which has a number of physiological functions, such as bone
development, placental calcium transport and cellular growth and development
(Martin et al., 1997
). In sea
bream (Sparus auratus L.), PTHrP has been detected in several tissues
and plasma by radioimmunoassay using antisera raised against the human peptide
(Danks et al., 1993
;
Devlin et al., 1996
) and, more
recently, the sea bream peptide (Rotllant
et al., 2003
). PTHrP has also been found in several other fish
species (Ingleton and Danks,
1996
; Danks et al.,
1998
; Trivett et al.,
1999
,
2001
). In addition, hormones
such as calcitonin (Wagner et al.,
1997
), growth hormone (Flik et
al., 1993
), vitamin D (Sundell
et al., 1992
) and cortisol
(Flik and Perry, 1989
) are
also known to be involved in the calcium balance of fish.
Sea bream is a euryhaline marine teleost that is important for
Mediterranean aquaculture. The intensive culture of this species leads to a
high number of morphological malformations, which typically result in growth
arrest, increased stress sensitivity and an increased incidence of disease
outbreaks (Andrades et al.,
1996; Carrillo et al.,
2001
). Improvement of our understanding of calcium regulation is
of paramount importance in improving proper development and growth of this
species in aquaculture settings.
We investigated calcium regulation after long-term exposure to limited calcium availability. The calcium balance of the fish was monitored through assessment of whole-body calcium and phosphorus content, plasma calcium levels and the relationship between calcium and phosphorus accumulation. In this context, we addressed hypercalcemic endocrine factors, viz PTHrP and cortisol, and investigated their relationship with calcium availability.
The experiments were achieved under controlled laboratory studies where sea bream were exposed to dilute seawater (hypocalcic values of 0.7 mmol l-1) and/or a calcium-deficient diet for prolonged periods of time.
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Materials and methods |
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Experimental set-up
To conduct the experiments, the required number of fish was randomly
selected from the stock group and transferred to six identical 60-litre round
tanks and left to acclimate. After one week, the salinity was lowered from
control salinity (34 10.5 mmol l-1 calcium) to test
salinity (2.5
0.7 mmol l-1 calcium) by continuous
flow-through with demineralized water, and the diet was gradually changed from
the control pellets (Trouvit) to the test pellets (Hope Farms, Woerden, The
Netherlands). The calcium-deficient and -sufficient diets were identical in
appearance (shape and colour). Although we observed temporary loss of appetite
when switching from control to diet pellets, feeding was resumed to comparable
levels after three days. This potential problem was addressed by keeping the
control diet fish group on a low diet regime (0.51% food of the total
mass) during the adaptation time to the new diet.
In the first experiment, five groups (AE) of sea bream (start mass,
17.4±4.6 g; N=20 per group; protandrous fish; not sexually
mature) were used. Group A is designated the control group (34,
control diet). The following test groups were included: group B (34
,
calcium-sufficient diet), group C (34
, calcium-deficient diet), group
D (2.5
, calcium-sufficient diet) and group E (2.5
,
calcium-deficient diet). The fish were exposed to experimental conditions for
six weeks and were fasted for 24 h before sampling. After three weeks
(t=1), all fish were weighed and 10 fish were euthanized with
2-phenoxyethanol (1:100; Sigma-Aldrich, St Louis, MO, USA), freeze-dried until
constant mass was reached and subsequently dissolved in concentrated nitric
acid (70%; 1 ml g-1 dry mass; Sigma-Aldrich) for mineral analyses.
Vials were carefully capped to avoid evaporation of the digest and the samples
were stored at 4°C. For the second sampling period [after six weeks
(t=2)], this procedure was repeated with the remaining fish
(N=10).
For the second experiment, the fish (N=24 per group) were exposed to experimental conditions for up to nine weeks; sampling took place after three (t=1), six (t=2) and nine (t=3) weeks. At each sampling time, eight fish were randomly selected, euthanized and weighed. Blood was taken from the caudal veins using 1 ml tuberculin syringes, rinsed with Na+-heparin (Leo Pharma, Weesp, The Netherlands; 5000 U ml-1) and diluted five times with demineralized water. Blood thus collected was centrifuged at 13 600 g for 10 min. Plasma was stored at 20°C.
Whole-body mineral concentrations
The nitric acid digests of fish were diluted 1000x with demineralized
water, and whole-body calcium and phosphorus were measured by Inductively
Coupled Plasma Atomic Emission Spectrophotometry (ICP-AES, Plasma IL200;
Thermo Electron, MA, USA). Mineral concentrations (µmol l-1) of
the digests were assessed, and content calculated and expressed as µmol
g-1 dry mass, based on digest total volume and fish dry mass.
In addition to calcium and phosphorus accumulation rates (µmol h-1), the correlation between the net accumulation of calcium and phosphorus was also calculated. Also, the relationship between mass and whole-body calcium (µmol) was determined and the so-obtained formula of this relationship was used to calculate the whole-body calcium levels of the second sampling group at t=1. Data of the measured whole-body calcium at t=1 and t=2 and the calculated data of the second group at t=1 were then pooled in full logarithmic plots of the relationship between mass and whole-body calcium at different calcium-limiting conditions. This was also done for the relationship between mass and whole-body phosphorus.
Plasma parameters
Plasma Ca2+ (µmol l-1) concentration was measured
with a Stat Profile pHOx plus analyser (Nova Biomedical, Waltham, MA, USA).
Plasma osmolality was measured using a cryoscopic osmometer (Gonotec Osmosat
030, Berlin, Germany) and expressed in mOsmol kg-1, and plasma
total calcium was measured with a calcium kit (Roche, Mannheim, Germany).
Plasma cortisol was measured by radioimmunoassay (RIA) as described by Arends
et al. (1999), and plasma PTHrP
was measured according to Rotllant et al.
(2003
).
Statistical analysis
All data were tested for significance by one-way analysis of variance
(ANOVA), followed by either Dunn's multiple comparison post test
(non-parametric) or the Bonferroni t-test (parametric), where
appropriate. Significance was accepted when P<0.05. All values are
expressed as means ± standard deviation (S.D.). Correlation
regression between two groups was determined with power function. Because no
variation was found in the results of the various parameters between three,
six and nine weeks, the data for each parameter were pooled to one data set
per group.
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Results |
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Whole-body calcium and phosphorus content
Net calcium and phosphorus fluxes in µmol h-1
(Fig.2A) and the correlation
between calcium and phosphorus accumulation rates
(Fig. 2B;
R2=0.92, N=16, P<0.01) demonstrate
that net calcium and phosphorus accumulation follow the same pattern, although
phosphorus availability was never limited under the experimental conditions.
The highest accumulation was observed in the control group (group A);
significantly lower net calcium and phosphorus accumulations were seen in the
test groups where calcium was limited (groups C and D). Group E exhibited the
lowest net calcium and phosphorus accumulation. The fish performed equally
well on control and calcium-sufficient pellets (groups A and B).
|
A logarithmic plot of the relationship between whole-body calcium and body
mass (M) shows a strong positive correlation
(Fig. 3A; R2=0.84, N=25, P<0.01). For the
control fish (group A), the relationship is described by the power function
Q=158.29xM1.27
(Table 1), where the calculated
slope of regression (1.27) reflects the rate of calcium accumulation
(Q; µmol) in the fish (Flik et al.,
1985,
1993
). The test groups show
lower power values (plots not shown), with comparable regressions in group B
and group D. The two groups exposed to calcium-deficient diet (groups C and E)
expressed the weakest slopes of regression. Overall, the power function
decreased with lower calcium availability.
|
|
Similar power functions were made for the relationship between whole-body phosphorus and body mass (Fig. 3B;R2=0.88, N=24, P<0.01). The regression slopes are comparable with the slopes that were found for the relationship between calcium and body mass, with the steepest slope in the control group (Q=279.81xM1.06) and lower phosphorusbody mass regression slopes at calcium-limiting conditions (Table 1).
Plasma parameters
In contrast to plasma total calcium levels, which differ significantly in
groups where calcium is limited in any way (groups CE), plasma ionic
calcium is strictly regulated except when fish are fed a calcium-deficient
diet and exposed to calcium-limited water
(Fig. 4). Under these
conditions, plasma ionic calcium did in fact decline significantly. Plasma
cortisol levels (Fig. 5A) are
low in controls (6.51±8.78 nmol l-1) and significantly and
chronically elevated in the test groups (up to 39.67±12.34 nmol
l-1) where calcium access was limited and a decline in total
calcium measured. Plasma PTHrP measurements show concentrations of
0.21±0.06 nmol l-1 (Fig.
5B) for the control group and a significantly higher plasma PTHrP
level of 0.30±0.11 nmol l-1 and 0.32±0.12 nmol
l-1 in groups C and D, exposed to either a calcium-deficient diet
or a low salinity, respectively. Group E, exposed to both 2.5 and a
calcium-deficient diet, expressed a comparable PTHrP level as the control
group.
|
|
For the control group, the positive correlation between plasma PTHrP and plasma ionic calcium is shown in Fig. 6. For PTHrP and total calcium, no such relationship was found (plot not shown). Also, for the test groups, significant correlations were absent.
|
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Discussion |
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Whole-body calcium
With respect to the calcium balance, prolonged exposure to diluted seawater
(2.5, which is a hypocalcemic medium) and a calcium-deficient diet
results in growth arrest in sea bream. This phenomenon has been described for
several other teleost species (Flik et
al., 1986
; Morgan and Iwama,
1991
; Woo and Kelly,
1995
; Sampaio and Bianchini,
2002
). Interestingly, the apparent growth arrest allows the fish
to maintain plasma calcium balanced at a level that ensures their survival for
prolonged times. Apparently, the calcium stores realised under control
conditions have a significant buffer capacity. We calculate, for a 50 g sea
bream, a total calcium content of 29.2 mmol under control conditions and of 16
mmol when water and diet are low in calcium. This indicates a 42% decrease in
the total calcium pool. Such drastically lower calcium content may be possible
only in aquatic vertebrates.
Plasma calcium
Ionic calcium levels are strictly regulated and fish are able to maintain
these physiologically important free calcium levels when calcium availability
is reduced in the diet and/or the medium. However, when calcium availability
is strongly reduced in both of the external calcium sources (the diet and the
medium), a slight but significant decrease in ionic calcium is observed. The
strict control of ionic calcium means that the calcemic regulation system must
be able to react swiftly on variable external calcium availability. A positive
correlation between the hypercalcemic hormone PTHrP and ionic calcium is
indeed found. This indicates that PTHrP is involved in the calcemic endocrine
control of plasma calcium balance in fish. Total calcium is not as tightly
regulated as ionic calcium by the calcemic control mechanisms, which means
that larger variations in plasma total calcium concentration are found,
indicating a change in binding protein level compared with the control group.
Indeed, no positive relationship between plasma total calcium and plasma PTHrP
is found here.
Calcium and phosphorus accumulation
The positive correlation found between body mass and whole-body calcium is
not affected by severe and chronic decreases in external calcium availability.
A similar relationship was found between body mass and whole-body phosphorus
for all experimental conditions. This is remarkable, because the experimental
conditions were focussed on calcium-limiting conditions, with phosphorus
concentrations unaffected. Since the phosphorus concentration in seawater is
very low, fish must depend on their diet for phosphorus, which they accumulate
at the same rate as that for calcium (Roy
and Lall, 2003). Yet, we have demonstrated that phosphorus
accumulation is impeded under conditions of low calcium availability
(Vielma and Lall, 1998
;
Chavez-Sanchez et al., 2000
).
Indeed, intestinal adsorption of phosphorus has been shown to be coupled to
calcium adsorption in a variety of vertebrates
(Mol et al., 1999
). These
studies mainly focus on the relationship between calcium and phosphorus in
relation to availability in diet and or medium and subsequently growth. In the
present study, we observed growth arrest under limiting calcium
concentrations. Since most of the whole-body calcium and phosphorus is
incorporated in bone and scales as calcium phosphate and calcium carbonate
complexes, growth arrest due to calcium-limiting conditions apparently also
leads to a subsequent decrease in net phosphorus influx.
PTHrP and cortisol
So far, only limited information is available on plasma PTHrP in sea bream.
Danks et al. (1993) measured
PTHrP in sea bream plasma and found 12.43±1.48 pmol l-1.
Here, we present PTHrP values of 0.21±0.06 to 0.32±0.12 nmol
l-1. These values are in line with the values reported by Rotllant
et al. (2003
), where, using
the same RIA as in this study, PTHrP values of 2.5±0.29 ng
ml-1 (0.61±0.07 nmol l-1) in 100150 g fish
were found. The lower values reported may well be caused by a lower
immunoreactivity of the heterologous antisera with fish PTHrP, explained by
different amino acids in the human N-terminal PTHrP sequence compared with
fish consensus (discussed by Rotllant et
al., 2003
).
The plasma PTHrP levels in the two groups that were exposed to either a calcium-deficient diet or a diluted medium show a significant increase compared with the plasma PTHrP level of the control group. However, when calcium was limited in both diet and medium, plasma PTHrP level did not increase when compared with the control fish. A possible explanation for this is that the results show that, although decreased, growth is continuing in the groups in which the fish still had access to a natural calcium source, either in the diet or medium. For this growth, a positive net calcium accumulation is required (which may well be supported by a hypercalcemic action of PTHrP), which is supported by our results. On the other hand, in the fish in group E, growth arrest occurs during the experiment. The net calcium accumulation in this group was 4.5-fold lower compared with the control group and 23-fold compared with the other test groups. Under their apparent growth arrest, no net calcium influx for skeletal formation is required. Apparently, the calcemic endocrine system successfully controls blood plasma calcium levels to a level that ensures proper physiology and survival of the fish.
Cortisol values are approximately two times higher in the 2.5 group
and 34 times higher in the calcium-deficient diet groups than in the
control group. Although significantly higher, these values still do not exceed
the basal level documented for this species, indicating that the fish were not
stressed. Arends et al. (1999
)
measured basal cortisol levels of 25 nmol l-1 in sea bream. These
values are in the same range as the basal levels in our experiment. It has
been shown before that subtle differences in basal cortisol levels could
account for changes in osmolarity, Na+/K+-ATPase
activity and plasma calcium levels (Metz
et al., 2003
). Flik and Perry
(1989
) demonstrated increased
cortisol secretion during hypocalcemic stress in freshwater rainbow trout,
inducing the uptake of calcium ions from the water by regulating the
Ca2+ pumps in the gills. Also, elevated plasma cortisol levels have
been shown to play a role in hypo-osmotic adaptation. Mancera et al.
(1994
) showed increased
cortisol levels in sea bream after transfer from 39
to brackish water
of 7
. The results reported here are corroborated by these early
findings.
In the present study, we have demonstrated that sea bream can cope well with limited calcium availability in either diet or medium. The fish continued to grow, and upregulated hypercalcemic hormones, PTHrP and cortisol, allow the fish to maintain the physiologically important ionic calcium level constant.
In the case of limiting calcium availability in both external calcium sources, growth arrest occurs in sea bream, and whole-body calcium level can be so maintained at such a level that no large net calcium accumulation is needed for skeletal formation. The relatively small net calcium accumulation rate that is still achieved by the fish can thus be used to maintain plasma calcium balance in such a way that it ensures the survival of the fish for a prolonged period of time.
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Acknowledgments |
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