Low temperature X-ray microanalysis of calcium in a scleractinian coral: evidence of active transport mechanisms
Analytical Electron Microscopy Laboratory, Department of Zoology, La Trobe University, Bundoora, Melbourne. Victoria, 3083, Australia
* Author for correspondence (e-mail: zooam{at}zoo.latrobe.edu.au)
Accepted 13 August 2002
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Summary |
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Key words: Low temperature, X-ray microanalysis, calcium, mucus, coral, Galaxea fascicularis, active transport Donnan equilibria, frozen-hydrated
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Introduction |
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In scleractinian corals, the ability to concentrate Ca at or near the site
of mineralisation necessitates migration of Ca2+ ions from the SW
across the epithelial layers. This may be achieved either directly from the
surrounding SW or via the coelenteron, which maintains direct contact
with the external SW environment via the mouth of the polyp. To date,
much of the research upon calcification in scleractinian corals has involved
the use of metabolic and enzymatic inhibitors and their effects upon rates of
45Ca deposition into the skeleton
(Marshall, 1996;
Tambutté et al., 1995
,
1996
). These studies have
indicated the involvement of an active transport process in the movement of
Ca2+ across the coral epithelia. Whilst it is probable that active
Ca2+ transport occurs in the aboral epithelia, immediately adjacent
to the skeleton (McConnaughey,
1995
; Tambutté et al.,
1996
), the mechanism of Ca2+ transport across the
outer, or oral, epithelium is less certain.
The mode of Ca2+ movement across the oral epithelium of
scleractinian corals has been variously reported to be both active
(Wright and Marshall, 1991)
and passive (Bénazet-Tambutté
et al., 1996
). The former studies were made upon sheets of
isolated epithelia from two scleractinian corals
(Wright and Marshall, 1991
)
and the latter upon isolated tentacles from a heliofungid scleractinian coral
and an anemone
(Bénazet-Tambutté et al.,
1996
). Since the evidence from these two investigations is
contradictory, we have attempted to measure in situ transepithelial
Ca concentrations in an effort to further elucidate the nature of
Ca2+ transport across the oral epithelium. This investigation was
prompted by previous studies on coral mucus (Marshall and Wright,
1991
,
1995
) and the realisation that
mucus could play a major role at the diffusive boundary layer of coral
epithelia. Such boundary layers have been suggested to have a marked influence
on ion transport in several ion-transporting epithelia (reviewed by
Shephard, 1989
;
Verdugo, 1990
;
Lichtenberger, 1995
;
Werther, 2000
).
Using a technique for preparing bulk, frozen-hydrated coral samples similar
to that described by Clode and Marshall
(2002), we have been able to
quantitatively determine element concentrations within distinct morphological
regions in the scleractinian reef coral Galaxea fascicularis. This
has allowed us to identify sites of calcium accumulation and to suggest
possible modes of Ca2+ transport. These bulk, frozen-hydrated
preparations are highly suitable for quantitative elemental analyses of
specialised and unspecialised cell types, distinctive cellular features and SW
compartments (coelenteric cavities) using low temperature X-ray microanalysis
(Marshall, 1987
,
1998
).
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Materials and methods |
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X-ray microanalysis
The basic method of X-ray microanalysis of frozen-hydrated bulk samples has
been described previously (Marshall,
1980a,
1987
,
1998
;
Marshall and Xu, 1998
) and is
a well established technique (Echlin,
1992
). Polyps were fractured under LN2 and small,
suitable pieces mounted in a modified sub-stage, so that the plane of the
fractured surface ran parallel to the stage. Strips of indium foil were
wrapped around the sample to improve conductivity and to provide flexible
compression when clamped in the vice-like sub-stage. Fractured preparations
were etched at -92°C before being coated with 200 Å aluminium in a
Cryo-Preparation System (CT 1500; Oxford Instruments). Samples of standard SW
in hollow rivets were frozen by plunging into liquid propane (-190°C). The
rivets were mounted in an appropriate substage and the samples were fractured
and coated in the cryo-preparation chamber. Both etched and unetched samples
were analysed.
Selected area analyses were conducted in a JEOL JSM 840A scanning electron
microscope (SEM) fitted with a Link exL X-ray analyzer and LZ5 detector.
Analyses were carried out with a Be window in place, for a period of 100 s
livetime, at 15 kV and a beam current (measured by a Faraday cup) of
2x10-10 A, on a custom built LN2-cooled stage
maintained at -174°C. Areas of analysis were no less than 25
µm2. Selection of regions for analysis, where local surface tilt
angle was close to 0°, was aided by using backscattered electron images,
as described by Marshall
(1981).
Spectra from selected area analyses were processed to yield peak integrals
by linear least-squares fitting to library peaks and quantitative data were
calculated using the PhiRhoZed model for matrix corrections
(Marshall, 1982;
Marshall and Condron, 1987
).
The standards used were polished microprobe standards (Biorad) of pure
elements or minerals of well-defined composition. For mucocyte analyses, C and
H concentrations were fixed at 10% and N at 3.3%, based on the elemental
composition of a 20% aqueous solution of generalised protein. Protein
composition was according to Engström
(1966
). Oxygen was calculated
by difference. For SW analyses (standard SW and SW compartments of polyps), H
was fixed at 10%, O was calculated by difference and C and N were omitted from
the calculations.
Because the highly mineralised coral polyps had to be fractured under
LN2 external to the cryo-preparation chamber associated with the
microscope, superficial frost had to be sublimed in order to visualise any
morphological features. This resulted in surface etching, which compromises
the accuracy of X-ray microanalytical data
(Marshall, 1981). Therefore,
the results of SW analyses were standardised by reference to Cl concentration
of SW at 33% salinity (Rankin and
Davenport, 1981
), since preliminary experiments indicated that Cl
concentrations in the different SW compartments analysed were not
significantly different from those of standard SW samples. Similarly, no
significant differences (P<0.05) were detected for any elements
between etched samples of standard SW, after analyses were standardised, and
unetched samples of standard SW in which analyses were not standardised. The
results from X-ray microanalyses of mucocytes were ratioed to S and expressed
as %S, because S was the predominant and invariable (P=0.99) element,
and because the other elements present are primarily cations, presumed to be
involved in charge neutralisation of sulphate groups.
Strontium analysis was based on the use of the Sr L X-ray spectral
lines. These correspond in X-ray energy almost exactly to the M lines
of tungsten X-rays. It was necessary, therefore, to correct for the extremely
small amount of tungsten deposited on the sample during evaporation of Al from
the tungsten filament. This was particularly important for the analysis of Sr
in SW compartments. Since the quantity of tungsten deposited was constant
under the same conditions of evaporation, the correction consisted of the
subtraction of a measurement of Sr concentration from a frozen sample (NaCl
solution), that did not contain Sr, from the Sr concentrations obtained from
the coral samples.
The analytical depth resolution at 15 kV is estimated to be approximately 2
µm and the lateral resolution similar
(Marshall and Condron, 1985).
Thus the volume from which X-rays are detected is roughly approximated by a
sphere of radius 1 µm. Results were expressed as mean ± standard
error of the mean (S.E.M.) and all statistical tests (both one-way analysis of
variance, ANOVA, with post-hoc analysis using Student's
t-test and Wilcoxon 2-group non-parametric test) were performed using
the computer software package JMP (SAS Institute Inc.). N is the
number of analyses, derived from 12 midday polyps and 4 midnight polyps.
Corallite samples
Soft tissue was removed from G. fascicularis polyps by submersion
in 5 mol l-1 NaOH at 60°C for 20 min. The resultant corallites
were rinsed in running water for 25 min and twice in distilled water for 10
min, before being infiltrated with 1000 mosmol kg-1 NaCl in 15%
polyvinyl-pyrrolidone (PVP) for 40 min. Preparations were then frozen in
liquid propane and stored as above. For analysis, PVP-infiltrated corallites
were fractured, etched and coated as described for frozen-hydrated polyps.
Digital linescans across the skeletal/NaCl-PVP interface were collected at 15
kV and a beam current of 2x10-10 A with a Be window in place.
The linescans recorded counts from an X-ray energy window covering the Ca
K peak (3.663-3.743 keV) and a region of background (3.423-3.503 keV)
at the low energy side of the peak. Subsequent to acquisition, the linescans
were arithmetically processed to yield linescans of peak-background/background
ratios.
Freeze-substituted sections
Frozen G. fascicularis polyps were freeze-substituted in 10%
acrolein in ether and embedded in araldite as described by Marshall
(1980b). Soft tissue was
dissected from skeleton, re-embedded and sectioned according to Marshall and
Wright (1991
). Sections were
stained with an aqueous BaCl2 solution (10 mmol l-1) and
imaged on a JEOL 1200EX scanning transmission electron microscope at 80
kV.
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Results |
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X-ray microanalysis of seawater compartments
Quantitative X-ray microanalysis of the external SW layer and extrathecal
and internal coelenteric SW compartments of freeze-fractured, frozen-hydrated
and etched G. fascicularis polyps revealed significant differences in
element concentrations between these compartments and standard SW
(Fig. 5). The major elements
present in standard SW were Na, Mg, S, Cl, K and Ca
(Fig. 5). In polyps sampled
during daytime, sulphur was present in significantly higher concentrations
(P<0.05) in the external SW layer (60±9 mmol
kg-1 wet mass; N=33) than in standard SW (29±0.8
mmol kg-1; N=17). Potassium concentrations were
significantly higher in both the external SW layer (34±6 mmol
kg-1; N=33) and the extrathecal coelenteron (19±2
mmol kg-1; N=38) than in standard SW (11±0.6 mmol
kg-1; N=17) (P<0.0001 and P<0.01,
respectively). Sodium concentrations in the external SW layer (546±12
mmol kg-1; N=33) and extrathecal coelenteron
(549±10 mmol kg-1; N=38) were also significantly
higher than in standard SW (501±12 mmol kg-1; N=17)
(P<0.05 and P<0.01, respectively).
|
Significantly higher calcium concentrations were observed in all SW compartments of daytime-sampled polyps in comparison to standard SW. The calcium concentrations of the external SW layer (16±0.7 mmol kg-1; N=33), extrathecal coelenteron (22±1 mmol kg-1; N=38) and internal coelenteron (23±2 mmol kg-1; N=18) were all significantly higher (P<0.01 for external SW layer and P<0.0001 for both internal compartments) than the calcium concentrations of standard SW (12±1 mmol kg-1; N=17) (Fig. 5). In addition, SW within the coelenteric compartments contained significantly higher (P<0.001) calcium concentrations than the external SW layer. An example of this gradient in calcium concentration is shown as raw X-ray counts in Fig. 6, for a single polyp. The gradient is apparent without conversion of X-ray counts to concentration values and standardisation. Notably, the major elemental components of mucus (S, K and Sr) all decreased in concentration within SW compartments in an inward gradient across the polyp (Fig. 5).
|
A comparison of element concentrations between polyps frozen in the daytime and night-time revealed that the concentrations of both S and Ca were significantly higher (P<0.05) in the external SW layer during the day (Fig. 7A). While not significant, Sr also showed a trend of lower concentration in the external SW layer in polyps sampled at night-time. No significant differences (P>0.05) in Na, Mg or K were observed within the external SW layer between midnight and daytime samples (Fig. 7A). In the extrathecal coelenteron SW, Mg concentration was significantly higher (P<0.05) in polyps sampled at night-time. No significant differences (P>0.05) in Na, S, K, Ca or Sr concentrations were observed within the extrathecal coelenteron SW between midnight and daytime samples (Fig. 7B). Within the internal coelenteron SW, no significant differences (P>0.05) in concentration were detected between daytime and night-time collected samples for any element (Fig. 7C).
|
X-ray microanalysis of mucocytes
Since there were no significant differences between analyses of mucocytes
in polyps frozen in the daytime or night-time, the data were pooled. The
primary elements detected in mucocytes of G. fascicularis were Na, S,
Cl, K, Ca and Sr (Fig. 8), with
S being the predominant and invariable element. Sodium, Cl and Ca all
increased in concentration (relative to S) in an inward gradient, with the
lowest concentrations detected in mucocytes of the oral ectoderm and the
highest in mucocytes of the calicoblastic ectoderm
(Fig. 8). Concentrations
differed significantly (P<0.0001) between the oral and aboral
epithelium for all three elements. In addition, the amount of Ca detected in
mucocytes of the calicoblastic ectoderm was significantly higher
(P<0.0001) than the amount of Ca present in mucocytes from any
other cell layer. The two other primary elements, K and Sr, were concentrated
in particular epithelial layers. Potassium was significantly higher
(P<0.0001) in concentration in ectodermal mucocytes compared to
gastrodermal mucocytes, while the reverse was true for Sr
(P<0.0001) (Fig.
8). No other significant differences (P>0.05) were
observed.
|
The ratios of K and Ca relative to S were calculated from the analytical data for mucocytes from the oral ectoderm, oral gastrodermis and aboral gastrodermis. Actual differences in element concentrations evident in SW compartments were then compared with expected concentrations, calculated on the basis that SW compartments contained standard SW and mucus secretions from mucocytes located within the surrounding epithelial layer(s). This calculation is based on the simplistic assumption that the mucus and associated cations dissolve in the SW. The concentrations of K and Ca (relative to S) observed in the external SW layer were slightly higher than the predicted ratios of standard SW containing secreted mucus from oral ectodermal mucocytes (Table 1). However, K and Ca concentrations in the extrathecal coelenteron were considerably higher than the expected ratios for standard SW containing mucus secretions from the oral and aboral gastrodermal mucocytes (Table 1), assuming that the mucocytes from each layer contributed equally to mucus within the SW.
|
Line scans resolution test
X-ray counts for Ca across the skeleton/NaCl-PVP interface of appropriately
prepared G. fascicularis corallites, revealed that when analysing
features close to the CaCO3 skeleton by X-ray microanalysis, Ca
X-rays generated from the bulk of the skeleton are minimal and contribute
little to the Ca signal. From the skeletal surface, the Ca X-ray signal was,
as expected, high; but as the beam moved away from the skeleton and into the
NaCl-PVP solution, this signal rapidly decreased
(Fig. 9). Almost no Ca X-ray
signal was detected when the beam was focussed more than 1 µm from the
skeletal surface.
|
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Discussion |
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In the mucus layer, which is located within the external SW layer, there did not appear to be a detectable reduction in Cl concentration relative to SW. This was also determined in our preliminary experiments and has additionally been observed in freeze-substituted sections (A. T. Marshall and O. P. Wright, unpublished data). The reason for this is not obvious, but the markedly lower S concentration in the mucus layer, compared with mucus granules in mucocytes, indicates a considerable degree of swelling and a concomitant reduction in negative charge density. Any reduction in Cl concentration, compared to SW, as a result of Donnan equilibria may, therefore, be very small and possibly masked by analytical variation. We estimate that any errors in determining Ca concentration resulting from standardising measurements to a fixed Cl concentration, would be less that 10%. Such an error would not significantly affect the subsequent calculations or change our conclusions. The possibility that the standardisation procedure could generate an artefactual Ca concentration gradient was eliminated by showing that the gradient persists in the unprocessed Ca X-ray counts across a single polyp.
For the quantitative analysis of mucocytes, matrix correction factors were calculated on the basis of an assumed composition of 10% C and 10% H. The content of C and H will vary depending upon the ratio of protein to oligosaccharide side chains and the water content. Both of these ratios are unknown. However, at the extreme limit, if the former is assumed to be 1.5 and the latter 20%, then the composition will be approximately 18% C and 8% H. Substitution of these values in the calculation for matrix correction factors results in essentially no change in the measured concentrations of the other elements present, with the exception of O.
The volume of the sample from which X-rays are emitted under the analytical
conditions used approximates a sphere 2 µm in diameter
(Marshall, 1982;
Marshall and Condron, 1985
).
In the analysis of frozen-hydrated corals we measured Ca concentration in the
mucocytes within the calicoblastic ectoderm, which varies from 1-5 µm in
thickness (Clode and Marshall,
2002
) and is immediately adjacent to the CaCO3
skeleton. It was therefore necessary to demonstrate that the analyses could be
accomplished without detecting extraneous Ca X-rays from the skeleton. Line
scan data clearly indicate that any Ca signal generated by scattering of
electrons to the bulk of the skeleton was minimal, and that concentrations
measured in mucocytes close to the skeleton were not artefactual.
Calcium entry
A coral polyp is essentially a sac-like organism with the interior of the
sac communicating with the external SW via the mouth. However, water
exchange via the mouth is probably extremely small. Consequently, the
coelenteron can essentially be regarded as a sealed compartment with an
internal medium that is isolated from the surrounding SW
(Wright and Marshall, 1991;
Bénazet-Tambutté et al.,
1996
; Furla et al.,
1998
). Coupled with the low water permeability of the oral tissue
(Bénazet-Tambutté and
Allemand, 1997
), the ionic environment of the coelenteric cavities
may become highly modified by active transport of particular ions, thereby
generating electrochemical gradients across both the oral and aboral
epithelial layers.
Several authors have suggested that Ca2+ transport across the
oral epithelium is an active, transcellular process (Chalker,
1976,
1981
;
Wright and Marshall, 1991
;
Marshall and Wright, 1998
).
This is supported by our results, which describe significantly higher Ca
concentrations in the extrathecal and internal coelenteron in comparison to an
external SW layer, adjacent to the outer surface of the polyp, and to standard
SW. These findings are consistent with those of Wright and Marshall
(1991
), but inconsistent with
those of Weber (1973
) and
Bénazet-Tambutté et al.
(1996
), who suggested that
Ca2+ transport across the oral epithelium occurred via
simple, paracellular diffusion.
Wright and Marshall (1991)
measured Ca2+ transport across isolated oral epithelia from the
theca of the scleractinian coral Lobophyllia hemprichii and the
vesicles of Plerogyra sinuosa, whereas Bénazet-Tambutté
et al. (1996
) used the oral
epithelia from the tentacles of an anemone Anemonia viridis and an
anemone-like scleractinian coral, Heliofungia actiniformis. On the
one hand, Bénazet-Tambutté et al.
(1996
) found that
Ca2+ moved passively across the tentacle epithelium and that the
epithelium was permeable to Na+ and Cl-. On the other
hand, Wright and Marshall
(1991
) showed that
Ca2+ was transported actively across the oral epithelium of the
body wall. Oral epithelial preparations from L. hemprichii were
impermeable to Na+ (using the Ussing chamber protocol described by
Wright and Marshall, 1991
; O.
P. Wright and A. T. Marshall, unpublished data). The flux of Na+
was measured in one paired experiment ectoderm to gastrodermis and
gastrodermis to ectoderm and four unpaired experiments, each of
ectoderm to gastrodermis and gastrodermis to ectoderm. The radioactivity in
the cold half of the Ussing chamber remained, in each experiment, at little
more than background levels after 320 min incubation, and in one case after 10
h incubation. Typically, the hot side contained 180x103
d.p.m. ml-1 whereas the cold side contained
<0.5x103 d.p.m. ml-1. It seems that the oral
epithelia from the body wall and the tentacles may have different permeability
properties.
Mucus composition
Meikle et al. (1987)
reported that mucus in scleractinian corals was composed of a glycoprotein
chain with numerous side chains of sulphated oligosaccharides. This
observation is supported by X-ray microanalytical data from mucocytes in
freeze-substituted coral preparations (Marshall and Wright,
1991
,
1995
;
Marshall and Clode, 1998
) and
by our analyses of frozen-hydrated preparations. As mucins are large,
negatively charged molecules that are not covalently linked, condensation of
mucins into mucus granules requires neutralisation of the mutually repulsing
polyanionic (sulphated) charges (see
Verdugo, 1990
;
Bansil et al., 1995
). In G.
fascicularis, high concentrations of the cations K+ and
Sr2+ in mucus granules suggests that these cations are likely to
play a primary role in neutralising the polyanions of mucin molecules. While
Ca2+ concentrations may be high
(Warner and Coleman, 1975
;
Takano and Akai, 1988
;
Verdugo, 1990
) and play a
major role in neutralising polyanionic charges of mucins in some secretory
epithelia (see Verdugo, 1990
),
it appears that this role is reduced in coral mucocytes. The detection of high
Na and Cl concentrations in G. fascicularis mucocytes is difficult to
explain. High Na and Cl concentrations have previously been reported in mucus
granules (Sasaki et al.,
1983
), including those of G. fascicularis (Marshall and
Wright, 1991
,
1995
), yet the function of
their association with mucin polymers remains unexplained.
Mucus secretion and the distribution of Ca
In G. fascicularis, the layer of mucus covering the oral ectoderm
is of the order of 10-20 µm thick
(Marshall and Wright, 1995),
and we suggest that this layer may play an important role in ion transport.
Mucus layers are known to influence the distribution of ions at epithelial
surfaces (reviewed by Shephard,
1989
; Verdugo,
1990
; Lichtenberger,
1995
; Werther,
2000
), this being a consequence of the effect of the mucus on
diffusion rates of ions and also of the participation of the constituent
negatively charged polyanions in Donnan equilibria.
Extracellular mucus may be regarded as forming a Donnan matrix, the
interactions of which are complex and not readily predicted
(Comper and Laurent, 1978). As
pointed out by Gupta (1989
),
the extracellular mucus matrix is not a quasi-permanent matrix supporting an
absolute Donnan equilibrium, but is continually being secreted and
demonstrates a complex steady state Donnan situation. In general, it would be
expected that the concentration of counterions (to polyanions) would be
increased, relative to the surrounding medium, and the concentration of
coanions would be reduced. Furthermore, most extracellular polyanions show a
strong preference for K+ over other cations, including
Ca2+ and Na+, although Na+ may be in much
higher concentration in extracellular fluid
(Scott, 1989
).
The distribution of Ca and K in the external SW layer and the extrathecal
coelenteron SW may be the result of a combination of Donnan equilibria and
transport across the oral epithelium. However, the ratios of Ca and K to S in
the external SW layer were very close to those measured in mucus granules
within mucocytes. This suggests that, unlike many secreted mucins
(Gupta, 1989), there is not an
exchange of counterions (eg. Ca2+ from mucin with Na+
from external medium) during the secretion process. Ion activities in mucoidal
solutions may be lower than in SW because the dissociation constants of
counterions with polyanions may differ from their constants in free solution
(Gupta, 1989
). However,
measurements of Ca2+ concentration with a mini ion-selective
electrode on the surface of the oral epithelium (A. T. Marshall and P. L.
Clode, unpublished data) are consistent with the concentration measurements
obtained by X-ray microanalysis. [The Ca2+ concentration measured
by ion-selective electrode in the SW layer at the surface of the epithelium in
the light (14.5±0.7 mmol l-1; N=3) was always
higher than in standard SW (11.3±0.3 mmol l-1;
N=3)]. Since the Ca2+ concentration measured by this type
of electrode is directly proportional to activity, it follows that there can
be no significant reduction in activity due to binding to the polyanion, when
compared with SW.
The establishment of a mucus layer at the SWoral ectoderm interface
may facilitate the uptake of Ca2+ into the oral ectodermal cells,
with Ca2+ able to exist in higher concentrations in a Donnan state
within this mucus matrix, in comparison to the normal SW environment. At
night-time when calcification rates fall
(Marshall, 1996), the demand
for Ca2+ is reduced and the presence and influence of this mucus
layer at the apical surface of the oral ectodermal cells, also declines. This
is indicated by lower S (indicative of mucus) and Ca concentrations detected
within the external SW layer in polyps sampled during night-time. A reduction
in the secretion of mucus and mucus-lipids at night time has also been
observed in the corals Acropora acuminata and Acropora
variabilis (Crossland et al.,
1980
; Crossland,
1987
). The K concentration in the external SW layer could also be
expected to be reduced at night-time. Failure to observe such a reduction can
be attributed to the presence of one, possibly anomalous, measurement of a
very high K concentration out of a total of four. It seems probable that a
greater number of analyses would have shown a reduction in K concentration
concomitant with the reduced S and Ca concentration.
Conclusions
The effects of an external mucus layer upon the microenvironment of the
oral ectodermSW interface and its effect upon the rates of ionic uptake
and exchange have not been previously recognised. Consideration has only been
given to the boundary layer in relation to O2 diffusion
(Kuhl et al., 1995;
Gardella and Edmunds, 1999
;
De Beer et al., 2000
). The
presence of a mucus layer within an external SW compartment on the surface of
the oral ectoderm may facilitate the maintenance of a Ca2+
concentration next to the ectodermal cells that is higher than that in
standard SW. This in turn may favour a high rate of entry of Ca2+
into the ectodermal cells during daytime. At night-time, calcification rate is
markedly reduced, but calcification still occurs
(Marshall, 1996
;
Marshall and Wright, 1998
).
Thus, it is not surprising that a high Ca concentration persists in the
internal SW coelenteric compartments. A continual presence of elevated Ca
concentration, detected within internal coelenteric compartments in corals
sampled during both daytime and night-time, suggests that maintenance of high
Ca concentrations within internal SW compartments is independent of the
formation of the mucus layer and Donnan state in the external SW layer. The
latter, however, may affect the rate of transport across the epithelium.
Certainly, Ca concentration appears to be limiting for calcification rate
(Marshall and Clode, 2002
). In
addition, it appears that mucus secretion into the coelenteric cavities is
minimal and that a substantial mucus secretion does not normally persist in
these internal compartments. This is indicated by the concentrations of S
detected within coelenteric cavities being only slightly higher than those of
standard SW. From this, it appears that mucus is not responsible for
establishing the higher Ca concentrations detected within internal SW
compartments. It is likely, therefore, that active transport mechanisms are
involved in the movement of Ca2+ across the oral epithelium,
resulting in the accumulation of significant amounts of Ca within internal SW
compartments.
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Acknowledgments |
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