Journal of Structural Biology 173 (2011) 99–109
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Journal of Structural Biology
journal homepage: www.elsevier.com/locate/yjsbi
Calcareous sponge biomineralization: Ultrastructural and compositional
heterogeneity of spicules in Leuconia johnstoni Carter, 1871
Christophe Kopp a, Anders Meibom a, Olivier Beyssac b, Jarosław Stolarski c, Shakib Djediat d,
Jakub Szlachetko e, Isabelle Domart-Coulon f,*
a
Muséum National d’Histoire Naturelle, Laboratoire de Minéralogie et Cosmochimie du Muséum (LMCM), UMR 7202, Case Postale 52, 61, rue Buffon, 75005 Paris, France
Institut de Minéralogie et de Physique des Milieux Condensés (IMPMC), UMR 7590 CNRS-IPGP-Universités Paris 6 & 7, Campus Boucicaut, 140 rue de Lourmel, 75015 Paris, France
Institute of Paleobiology, Polish Academy of Sciences, Twarda 51/55, 00-818 Warsaw, Poland
d
Muséum National d’Histoire Naturelle, Département RDDM, USM 505 Case Postale 39, 57 rue Cuvier, 75005 Paris, France
e
European Synchrotron Radiation Facility, X-Ray Microscopy Beamline ID21, B.P. 220, 38043 Grenoble Cedex, France
f
Muséum National d’Histoire Naturelle, Département Milieux et Peuplements Aquatiques, UMR 7208 MNHN-CNRS-IRD-UPMC Case Postale 26, Biologie des Organismes et des
Ecosystèmes Aquatiques (BOREA), 43 rue Cuvier, 75005 Paris, France
b
c
a r t i c l e
i n f o
Article history:
Received 12 May 2010
Received in revised form 12 July 2010
Accepted 17 July 2010
Available online 23 July 2010
Keywords:
Calcareous sponge
Biomineralization
Calcite
Ultrastructure
Composition
Spicule
a b s t r a c t
In contrast to siliceous sponge spicules, the biomineralization in calcareous sponges is poorly understood.
In particular, the existence of a differentiated central core in calcareous spicules is still controversial. Here
we combine high-spatial resolution analyses, including NanoSIMS, Raman, SXM, AFM, SEM and TEM to
investigate the composition, mineralogy and ultrastructure of the giant tetractines of Leuconia johnstoni
Carter, 1871 (Baeriidae, Calcaronea) and the organization of surrounding cells. A compositionally distinct
core is present in these spicule types. The core measures 3.5–10 lm in diameter and is significantly
depleted in Mg and lightly enriched in S compared with the adjacent outer layer in the spicule. Measured
Mg/Ca ratios in the core range from 70 to 90 mmol/mol compared to 125–130 mmol/mol in the adjacent
calcite envelope. However, this heterogeneous distribution of Mg and S is not reflected in the mineralogy
and the microstructure. Raman spectroscopy demonstrates a purely calcitic mineralogy. SEM examination of slightly etched spicules indicates an ultrastructure organized hierarchically in a concentric pattern, with layers less than 250 nm in width inside layers averaging 535 ± 260 nm. No change in
structural pattern corresponds to the Mg/Ca variation observed. AFM and TEM observations show a nanogranular organization of the spicules with a network of intraspicular organic material intercalated
between nanograins 60–130 nm in diameter. Observations of sclerocyte cells in the process of spiculogenesis suggest that the compositionally distinct core is produced by a sub-apical sclerocyte ‘‘founder
cell” that controls axial growth, while the envelope is secreted by lateral sclerocytes ‘‘thickener cells”,
which control radial growth.
Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction
Calcium carbonate spicules provide skeletal support for marine
calcareous sponges (Calcarea, Porifera). Although the shape, size
and spatial arrangement of spicules in the sponge body have traditionally been a very important taxonomic tool (Manuel et al.,
2003), the biomineralization processes by which they form are still
poorly understood. As a first step towards a better understanding
of calcareous sponge spicules formation, it is necessary to characterize potential spatial variations in their composition and ultrastructure. Such observations could provide indications on the
* Corresponding author. Fax: +33 1 40 79 37 71.
E-mail address: icoulon@mnhn.fr (I. Domart-Coulon).
1047-8477/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jsb.2010.07.006
biological control of the organism, at the cellular level, during
the biomineralization process.
Calcareous sponge spicules are extracellular biominerals
formed in the mesohyl of the sponge body between the pinacoderm surface and the choanocyte filtration chambers. Scenarios
of formation have been proposed since the 1970s based on ultrastructural observations. It is currently believed that spicules form
in an intercellular cavity between several specialized spiculesecreting cells, the sclerocytes (Ledger and Jones, 1977), sealed
by septate junctions (Ledger, 1975). Growing spicules are enveloped by a thin organic sheath (Jones, 1967; Ledger, 1974; Ledger
and Jones, 1977), which later becomes enclosed in the network
of collagen connective fibrils positioning mature spicules in the
sponge mesohyl (Simpson, 1984; Sethmann and Wörheide,
2008). Rates of spiculogenesis have been assessed based on 45Ca
labeling and the incorporation of tetracycline fluorochrome,
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C. Kopp et al. / Journal of Structural Biology 173 (2011) 99–109
suggesting a 1–3% spicule turn-over in Clathrina cerebrum over
24 h in short-term aquarium experiments (Bavestrello et al.,
1993, 1994). Dynamics and growth pattern of calcareous spicules
were also studied in Sycon sp with calcein fluorescence labeling
(Ilan et al., 1996), revealing marked differences in growth rates
depending on spicule type, with slender monaxon deposition rate
increased by a factor 6 compared to curved monaxon.
Crystallographic studies and bulk analyses of their chemical
composition have revealed low-Mg calcite (Jones and Jenkins,
1970) behaving as a single-crystal (reviewed by Sethmann and
Wörheide, 2008). Ultrastructural observations of intact and fractured spicules showed a polycrystalline organization into nanogranular clusters and suggested organic matter intercalation
between small crystal domains (Sethmann et al., 2006). Aizenberg
et al. (1996b) have detected small amounts of intraspicular proteinaceous macromolecules (0.07–0.1 wt.%) characterized by a
content rich in asparagine and/or aspartic acid. At the microscale,
spicules are characterized by a pattern of successive concentric
layers (traditionally called ‘stratification’), which are observed
mostly in the larger triactine and tetractine types by alkaline or
acidic etching (e.g. Jones and James, 1972; Jones, 1970; Ledger
and Jones, 1991; Von Ebner, 1887). However, the existence of a differentiated central core in the calcareous sponge spicules has always been controversial. By comparison, it is well established
that a central organic axial filament is present in the siliceous spicules of Demospongiae and Hexactinellida sponge species (Uriz
et al., 2003; Uriz, 2006).
Building on Von Ebner’s 1887 spicule heating experiments and
Bütschli’s 1901 acid etching experiments, Minchin (1898) and then
Minchin and Reid (1908) obtained an axial residue stainable with
nigrosin or indulin from spicules decalcified with diverse acids,
suggesting the existence of an organic axial filament. However,
Jones (1967) (later confirmed by Ledger and Jones (1991)) observed that this organic axial filament was an artifact corresponding to remnants of the spicule organic sheath contracted during the
decalcification process. Etched spicules of e.g. giant triactines of
Leuconia nivea sometimes revealed central hollows (Jones and
James, 1972; Ledger and Jones, 1991), which could suggest a differentiated axial core. According to Von Ebner (1887), the axial portion of the spicules was ‘a region of less pure calcite compared to
the peripheral portion, incorporating impurities such as magnesium, sodium and sulfate ions’ and the stratification of spicule
was due to the ‘periodic deposition of less pure and more pure calcite’. Jones (1970) also suggested that varying Mg content might
explain the concentric lamination pattern. However, no significant
variations in the spatial distribution of Ca, Mg, Sr and S could at
that time be detected with the electron micro-probe along profiles
through transverse and longitudinal sections of rays of giant triactines of L. nivea, probably due to low spatial resolution (>1 lm) and
sensitivity (Jones and James, 1969). Based on the fact that the
smaller types of spicules of L. nivea and Amphiute paulini contained
less Mg than the larger ones (Jones and Jenkins, 1970), Jones (1970)
proposed the hypothesis of a potential heterogeneous distribution
of Mg in spicules, with the first secreted spicular material depleted
in Mg compared to the peripheral deposits. Recently Sethmann
et al. (2006) have used high-resolution TEM–EDX to detect nanometer-scale heterogeneity of Mg distribution in fractured spicules.
However, these observations were not related to micrometric features like core or concentric layers or to differences in cellular
activity.
Mineralogical heterogeneity was reported by Aizenberg et al.
(1996a, 2003) in the spicules of Clathrina sp (collected off Atlit, Israel) with a calcitic core enveloped by a thick layer of ACC, which
was covered by a thin calcitic layer. However, it was not clear if
such proposed mineralogical stratification was species specific or
widely distributed among calcareous sponges.
In this work we have mapped at high, sub-micrometric, spatial
resolution with SEM,1 AFM, NanoSIMS, SXM and Raman mapping
the structure and composition of giant tetractines of Leuconia johnstoni Carter, 1871. The main purpose is to examine compositional
variations of calcareous spicules in relation to their ultrastructure.
The ultrastructure of sclerocytes (spicule-secreting cells) and spicules was imaged with TEM and SEM, and a cellular model is proposed for axial growth (elongation) versus radial growth
(thickening) to explain the spatial variations in trace element distribution detected in the calcitic spicules.
2. Materials and methods
2.1. Biological material
Specimens of the calcareous sponge L. johnstoni Carter, 1871
(Baeriidae, Calcaronea, Calcispongia) were collected in the subtidal
zone under granite boulders (Fig. 1a) in South Finistère off Concarneau, France, between May 2008 and October 2009 (47°52N,
3°55W).
L. johnstoni is a littoral marine species living on sub-vertical
rock surfaces in wave exposed sites, with a distribution ranging
from the British Isles to the Channel coasts of France and the Gulf
of Biscay (Picton and Morrow, 2007). This small-sized encrusting
sponge (less than 50 mm diameter and 15 mm height) is composed
of compressed lobes fused at their base and bearing apical openings. The color may be white to beige and silt is often trapped between the lobes on the surface. The specimens were fixed either
with paraformaldehyde 3% or ethanol 70%, or were directly frozen
at 20 °C. Taxonomic determination of the specimens was confirmed based on examination of spicule nature and arrangement,
and by comparison to reference L. johnstoni from the MNHN collection of Porifera (microscopic preparations from the Borojevic Roscoff collection voucher C-1968-426, and from the Haeckel collection
voucher C-1968-678).
2.2. Morphological analysis of spicule types and orientation
To isolate spicules, small sponge fragments were bleached at
room temperature in 5% commercial sodium hypochloride solution
(NaClO) on a rocking table for 1 h to digest the organic matter. The
dissociated spicule suspension was then briefly rinsed three times
in tap water, rinsed once in deionized water and transferred to
absolute ethanol. The morphology of isolated spicules was observed in reflection with a stereomicroscope LEICA MZFLIII or in
transmission (in permanent araldite slide mounts) with a microscope LEICA DMRB. Images were acquired with a LEICA DC300F
camera (Leica, France) and processed with IM50 software. For
SEM observations, isolated spicules were deposited on a stub,
coated by gold (5 nm thick) and examined at 15 keV with SEM
TESCAN (model VEGA II LSU) or with SEM JEOL JSM-840 at the
Muséum National d’Histoire Naturelle (MNHN) (Paris, France).
Alternatively, sponge fragments were cryofractured in absolute
ethanol, and either critical-point dried by CO2 substitution or
lyophilized and then mounted on SEM stubs. Spicular arrangement
and cell–spicule interface were observed in the fracture area at
15 keV with the SEM JEOL JSM-840 A at the MNHN.
Calcareous sponge spicules are classified according to the number of rays (actines) that they possess. Thus, the different spicule
categories are monaxon – a term including monactine (one ray)
and diactine (two rays) – triactine (three rays) and tetractine (four
1
Abbreviations used: SEM, scanning electron microscopy; AFM, atomic force
microscopy; NanoSIMS, secondary ion mass spectrometer; TEM, transmission electron microscopy; EDX, energy dispersive X-ray spectroscopy; SXM, scanning X-ray
microscope; ACC, amorphous calcium carbonate.
C. Kopp et al. / Journal of Structural Biology 173 (2011) 99–109
101
Fig. 1. Macromorphology of Leuconia johnstoni. (a) Live specimen encrusting granite boulders in the tidal zone. (b) SEM micrograph of a sponge fragment cryofractured near
the apex of a lobe, indicating that the sponge is composed of an outer ectosome (ect) rich in large spicules and inhalant canals, covering the choanosome containing the
choanocyte filtration chambers (ch), the exhalant canals and a small atrial central cavity. Triactines and the basal triradiate system of giant tetractines (white arrows) are
disposed tangentially to the ectosome, whereas the apical ray of giant tetractines is directed internally, perpendicular to the sponge surface.
rays). Tetractines are built from three rays disposed like a triactine,
referred to as the basal triradiate system, with an additional ray
called the apical actine (Boury-Esnault and Rützler, 1997).
2.3. Compositional analysis
Small fragments of L. johnstoni were dried and embedded in
Körapox epoxy resin. They were then polished with diamond suspensions (particle sizes = 3 lm, 1 lm and 0.25 lm), revealing multiple sectional planes through rays. During this process, some
fracturing of the rays occurred, especially for the smallest types
of spicules, which are very fragile.
Following established procedures (Meibom et al., 2004, 2008),
the spatial distribution of Mg was mapped out in transverse and longitudinal sections of rays of giant tetractines of L. johnstoni with the
Cameca NanoSIMS ion micro-probe at the Laboratoire de Minéralogie et de Cosmochimie du Muséum (LMCM) of the MNHN. Briefly,
with a primary beam of O , focused to a spot-size of 200 nm on
the gold-coated surface of the sample, secondary ions of 24Mg+
and 44Ca+ were sputtered from the sample surface and detected
simultaneously in multi-collector mode with electron multipliers
at a mass-resolving power of 4500. At this mass-resolving power,
the measured secondary ions are resolved from potentially problematic interferences. Maps were obtained by rastering the primary
beam across a pre-sputtered surface. Magnesium concentrations
were calibrated against carbonate standard of known composition.
Sulfur content was mapped at high-spatial resolution in crosssection of a giant tetractine spicule with SXM operating in the Xray fluorescence mode at the X-ray Microscopy beamline ID21 of
European Synchrotron Radiation Facility (Grenoble, France) following procedures described by Cuif et al. (2003). The X-ray beam,
monochromatized by means of double-crystal (Si(1 1 1)) fixed exit
monochromator, was tuned to an energy just above the K absorption of sulfur. The Kirkpatrick–Baez mirror arrangement was employed to focus the X-ray beam down to size of 0.3 0.8 lm2.
The X-ray fluorescence spectra were recorded by HpGe detector
placed at 90° scattering angle. A 2D image was obtained by
point-by-point scanning of the sample across the focal point of
the beam, with typical exposure time of 150–300 ms per point.
2.4. Micro- and nanostructural analysis
Following NanoSIMS analysis, the gold was delicately removed
by polishing with a diamond paste of 0.25 lm particle size. The
samples were then slightly etched, during approximately 2 min
15 s, with solution of 0.1% formic acid (pH 3–4) and 2.5% glutaraldehyde, then rinsed in tap water and ethanol 70%, dried and coated
with gold (15 nm). Examinations of etched sections of giant tetractines were performed with SEM Tescan (model VEGA II LSU)
at the MNHN. Giant tetractines were recognized by their diameter
and/or the length of their rays. Microstructural organization was
not characterized for the other categories of spicules, especially
the smallest ones, because they were too fragile to resist polishing
or completely dissolved by the etching.
Nanostructural analysis was investigated with AFM performed
with a MultiMode Nanoscope IIIa (Digital Instruments, Veeco), following procedures described by Stolarski and Mazur (2005). Standard silicone nitride cantilevers were used for measurements in
tapping mode. Sections of giant tetractines polished as previously
described were examined after etching in 1% ammonium persulfate in McIlvain buffer (pH 8) for 10 min, followed by rinsing in
deionized water and drying.
2.5. Mineralogical analysis
For mineralogical analysis, spicules were prepared from sponge
specimens fixed by freezing at 20 °C and stored frozen at 20 °C
until use, in order to avoid crystallization of potential unstable ACC
phase. Isolated spicules were thawed just prior to Raman microspectroscopy. Raman maps were obtained from the surface of apical rays of giant tetractines freshly broken in a transversal way
before analysis. Spot analyses were carried out in three different
places on the surface of this ray: the center, the medium and the
extreme periphery (respectively noted (1), (2) and (3) on Fig. 6a).
Raman spectra were also acquired from the surface of minute tetractines, microdiactines, pugioles and triactines (data not shown).
All the spicules were placed on an aluminium foil-covered glass
slide. Raman maps and spectra were acquired using a Renishaw InVia Reflex micro-spectrometer at the Institut de Minéralogie et de
Physique des Milieux Condensés (IMPMC) (Paris, France). A
785 nm near-infrared diode laser (Renishaw) was focused on the
sample by a DMLM Leica microscope with a 50 (Numerical Aperture = 0.55) or a 100 (Numerical Aperture = 0.70) objective. The
Rayleigh diffusion was eliminated by edge filters and the signal
was finally dispersed using a 1800 lines/mm grating and analyzed
by a Peltier cooled RENCAM CCD detector. Before each session, the
spectrometer was calibrated with a silicon standard. For Raman
mapping we used the dynamic line-scanning mapping device
Streamline. See Bernard et al. (2008) for more details.
2.6. Histological and ultrastructural analysis of cell structures
For TEM, fragments of L. johnstoni sponges were fixed in 2% glutaraldehyde in modified Sörensen phosphate buffer (NaH2PO4 and
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C. Kopp et al. / Journal of Structural Biology 173 (2011) 99–109
2H2O–Na2HPO4 0.1 M adjusted to pH 7.6 and sucrose 0.6 M) with
20 mM CaCl2 and 0.055% w/v Ruthenium Red. Fragments were
cut into smaller pieces, which were partly decalcified by ascorbic
acid 2% for 24 h at room temperature. They were postfixed in 1%
OsO4 in modified Sörensen buffer and dehydrated through a
graded ethanol series. The pieces were embedded in Spürr resin.
Sections were cut with a Diatome 35° diamond (Ultracut microtome). Semi-thin sections were stained with solution of 1% toluidine blue, 1% borax in 70% ethanol and observed with a
microscope LEICA DMRB equipped with a LEICA DC300F camera
(Leica, France). Ultra-thin sections were counterstained with uranyl acetate 2% in 50% ethanol and were observed at 75 keV with
a Hitachi H7100 transmission electron microscope (TEM) equipped
with a digital CCD Hamamatsu camera at the MNHN.
3. Results
3.1. The different types of spicules of L. johnstoni
The skeleton of L. johnstoni (76% of the sponge total dry weight)
is composed of seven different categories of spicules: (1) large sagittal (giant) tetractines (Fig. 2a) with paired rays 220–820 lm long
by 25–100 lm maximum width, unpaired ray 180–480 lm long by
25–100 lm maximum width and apical ray 160–725 lm long by
20–100 lm maximum width; (2) sagittal triactines (Fig. 2b), with
paired rays 80–480 lm long by 10–35 lm maximum width and
unpaired ray 85–360 lm long by 10–30 lm maximum width; (3)
dagger-shaped small sagittal tetractines (Fig. 2c), termed pugioles
and characteristic of the order Baerida (Borojevic et al., 2002), with
paired rays 25–55 lm long by 10 lm maximum width, unpaired
ray 65–100 lm long by 10 lm maximum width and apical ray
75–95 lm long by 10 lm maximum width; (4) long, smooth, and
slightly curved diactines (Fig. 2d), 250–670 lm long by 15–
25 lm maximum width; (5) microdiactines 45–65 lm long by 3–
5 lm maximum width (Fig. 2e) ornamented by rows of minute
spines (Fig. 4d); (6) regular, minute sized, tetractines (Fig. 2e and
g) with rays of the basal triradiate system 10–20 lm long by 1–
2 lm maximum width and a short apical ray 2–3 lm long; (7)
long, very fine and straight monaxons located on the external surface near the oscules (Fig. 2f). These observations match the original description of the spicule types in the L. johnstoni species by
Carter (1871), with additional measurements. Each spicule ray behaves as a single-crystal in polarized light. In cryofractured specimens, the surface ectosome is differentiated from the internal
choanosome (Fig. 1b). The ectosomal skeleton of the sponge is
composed of tangential triactines and of the tangential basal trira-
diate system of giant tetractines (Fig. 1b). Their unpaired rays seem
to be all oriented towards the sponge substrate (data not shown).
The apical ray of giant tetractines is directed internally, perpendicular to the sponge surface (Fig. 1b). The choanosome is rich in
microdiactines, pugioles and a few minute tetractines.
In this work, we have focused our analyses on the giant tetractines of the sponge ectosome, because they were less fragile than
the other spicules and presented a surface more adequate for spatial analysis.
3.2. Microstructural concentric internal layers and nanostructural
composite organization of the spicules
Fig. 3 illustrates the microstructure of giant tetractines revealed
by slight acidic etching of polished sections with 0.1% formic acid
during 2 min 15 s. Giant tetractines are composed of fine concentric bands (Fig. 3a–c). Here, we define a band by the succession of a
furrow of etched material and a ridge of acid-resistant material (inset of Fig. 3b). The lamination is more pronounced in certain regions of the rays and these differences may result of a potentially
heterogeneous efficiency of the etching solution (artifact). In transverse sections layers less than 250 nm wide, referred to as primary
bands, are detected inside larger layers, referred to as secondary
bands. This is especially visible in the spicule center, where the layers are clearly ellipsoid (Fig. 3b). Average width of secondary bands
is estimated to 535 ± 260 nm (n = 136 measures taken from different regions of transverse and longitudinal sections of 14 different
rays). In longitudinal sections bands seem wider near the apex of
the ray than laterally (Fig. 3c).
A near rectilinear ridge crosses systematically the central region
of the rays in transverse sections and could define a symmetric axis
for the axial ellipsoid bands (Fig. 3b). This ridge extends in the
range of 12–33 lm.
At the nanostructural level, AFM results show that spicules consist entirely of an assemblage of nanograins ca. 60–130 nm in
diameter, separated from each other by spaces of a few nanometers (Fig. 4a and b). This nanogranular organization is also supported by observations with SEM of the conchoidal fracture area
of rays (Fig. 4c), in every spicule type. Complementary TEM examination of partially decalcified microdiactines (Fig. 4d–f) reveals
two kinds of organic material associated with spicules, both
stained by Ruthenium Red, indicating a content rich in proteoglycans: a very thin organic sheath enveloping the spicules (no more
than few dozen nanometers wide); a networked intraspicular organic material delineating small compartments of approximately
the same size as nanograins detected with AFM. These small com-
Fig. 2. The seven types of spicules composing the skeleton of Leuconia johnstoni. Optical images (a–e) illustrate various spicules at the same scale, after dissociation with
bleach from the sponge. SEM micrographs (f, g) of spicules in situ in the sponge body fragment near an oscule. (a) Giant sagittal tetractine, with indication of the terminology
for each ray. (b) Sagittal triactine. (c) Small tetractine, termed pugiole. (d) Long, smooth, thick and slightly curved diactine. (e) Small microdiactine (left) and minute tetractine
(right). (f) Long, very fine and straight monaxons located on the external surface (black arrow) between the abundant giant tetractines and large triactines. (g) Minute
tetractine embedded in the wall of an exhalant canal.
C. Kopp et al. / Journal of Structural Biology 173 (2011) 99–109
103
Fig. 3. Microstructural organization of rays from giant tetractines of Leuconia johnstoni, revealed by slight acidic etching with 0.1% formic acid (2 min, 15 s) indicating a
concentric lamination pattern. Large rectilinear scratches across the sections are polishing arctifacts. (a) Transverse section of a ray. (b) Detail of (a). The inset is a higher
magnification of the axial region of the ray. Bands, defined by a doublet furrow plus ridge, are clearly visible. White arrows indicate the position of a systematically centrally
located near rectilinear ridge. A primary banding is visible (black arrowhead) within the secondary bands of the axial region of the ray. (c) Longitudinal section of a ray. The
inset represents the whole ray for orientation and the square indicates the general location of the enlarged view.
Fig. 4. Nanostructural organization of spicules of Leuconia johnstoni. (a) AFM height mode and (b) amplitude mode image of the surface of a spicule in transverse section.
Spicules consist of nanograins ca. 60–130 nm in diameter separated from each other by a thin space of few nanometers. (c) SEM micrograph of the surface of a fracture
through a triactine ray revealing similar nanograins (inset) to those observed with AFM; organic remnants of the unbleached spicule envelope are visible. (d) SEM image of a
dissociated microdiactine ornamented by rows of minute spines (inset). (e and f) TEM micrographs of a partially decalcified microdiactine showing organic material stained
by Ruthenium Red proteoglycan fixative, forming the outer sheath and a networked intraspicular organic material delineating nanograins similar in size to those observed
with AFM. These nanograins may form a peripheral layer 100 to 250 nm wide (inset (e)).
partments can be seen forming a peripheral layer 100 to 250 nm
wide (Fig. 4e), which may correspond to the primary banding
increments reported for giant tetractines.
3.3. Trace element heterogeneity in the giant tetractines at
ultrastructural length scale
With the high-spatial resolution (200 nm) of the NanoSIMS
the distribution of Mg was mapped in transverse sections of five
rays, each belonging to a different giant tetractine (Fig. 5a illustrates two examples). Mg was also mapped in longitudinal section of a ray from a giant tetractine (Fig. 5b). In all spicules
analyzed in transverse section, the core of the ray is systematically depleted in Mg compared to the envelope of the ray
(Fig. 5a). The diameter of this Mg-depleted core, more or less
cylindrical, ranges from 3.5 to 8 lm and corresponds in longitudinal section to a central band (Fig. 5b). The Mg/Ca ratio in the ray
envelope averages approximately 125–130 mmol/mol, with fluctuations less than 10% and no periodicity (Fig. 5c and d). This ratio drops substantially in the ray core to reach values between 70
and 90 mmol/mol, which represent a diminution by 30% to 45%
compared to the envelope (Fig. 5c and d). With SXM operating in
the X-ray fluorescence mode a map of S distribution was obtained
from a transverse section of a ray from a giant tetractine (Fig. 5e)
showing an ellipsoid central ray core, 4.5 to 10 lm in diameter,
lightly enriched in S compared to the ray envelope. There is a
strong spatial correlation between the Mg-depleted area and the
S-enriched area in the core of spicular rays (compare Fig. 5a
and e). The extreme periphery of rays also appears to be enriched
in Mg and S (Fig. 5a and e), conceivably due to remnants of organic sheath and cells covering the spicule (Fig. 5f). Spatial variations in Mg/Ca ratio and S content are thus tightly regulated in
spicular rays and define a compositionally differentiated spicule
core.
104
C. Kopp et al. / Journal of Structural Biology 173 (2011) 99–109
Fig. 5. Spatially heterogeneous distribution of Mg (a–d) and S (e) in the rays of giant tetractines of Leuconia johnstoni. The axial region of the rays is depleted in Mg and
enriched in S. (a) NanoSIMS maps of Mg distribution from two different rays cut transversely. The inset is a map centered on the axial region of a ray. Red color code indicates
regions of high Mg/Ca ratios versus blue for low-Mg/Ca. (b) NanoSIMS map of Mg distribution in the axial region of a ray cut longitudinally. (c) Fluctuations of Mg/Ca ratio,
quantitatively expressed along the AB transect of (a). (d) Fluctuations of Mg/Ca ratio along the CD transect of (b). (e) SXM map of S distribution in a ray cut transversely. The
red arrow indicates the position of the sulfur enriched axial region (light blue color code). (f) SEM micrograph of a ray showing a conchoïdal fracture pattern. The ray is
enveloped by an organic sheath and by sponge cells (red arrow).
3.4. Uniform calcitic mineralogical composition of giant tetractines at
microstructural length scale
Raman spectra were acquired with a spot-size of 1 lm
whereas Raman maps were obtained with a step width of 1 lm
directly from the surface of an apical ray of a giant tetractine
freshly broken along a transversal fracture (Fig. 6a). Three representative spectra obtained from spot analysis of the center, the
medium part and the extreme periphery of this ray (respectively
noted (1), (2) and (3) on Fig. 6a) exhibit bands characteristic of
magnesian–calcite (Fig. 6b): two lattice modes at 156 cm 1 and
283 cm 1, t1 CO3 symmetric stretching at 1090 cm 1 and t4
antisymmetric stretching at 716 cm 1. These values are globally
similar to other synthetic and some biogenic magnesian–calcites
analyzed with Raman spectroscopy with similar Mg composition
in the range 70–130 mmol/mol (or 7 to 13 mol% MgCO3) (Bischoff
et al., 1985; Urmos et al., 1991). Raman mapping confirm these
observations and highlight the remarkable constancy of the frequency and full width at half maximum (FWHM) of these four
bands over the whole section of the ray (Fig. 6c–e). In addition,
the two lattice modes are systematically clearly observed over
the whole section. Altogether, this suggests that giant tetractines
of L. johnstoni are entirely composed of magnesian–calcite. No
amorphous phase, like ACC was detected. Similar observations performed for some smaller types of spicules: minute tetractines,
microdiactines, triactines and pugioles gave exactly similar spectra
of magnesian–calcite (data not shown). This strongly suggests that
the seven forms of spicules present in L. johnstoni are entirely composed of magnesian–calcite. The weak broad band observed near
1010 cm 1 (Fig. 6b) may be attributable to bicarbonate (HCO3 )
ions (Bischoff et al., 1985).
It is interesting to note that the fluctuation of the Mg content
between the core (70 to 90 mmol/mol, or 7 to 9 mol% MgCO3)
and the envelope (125 to 130 mmol/mol, or 12.5 to 13 mol%
MgCO3) of rays from giant tetractines was not detected by Raman
spectroscopy. Indeed, no significant differences in the Raman band
frequencies or FWHM were observed from the core to the outer
rim of the ray analyzed (Fig. 6c–e). It has been shown that fluctuations of 7 to 13 mol% MgCO3 are also not clearly detected with
Raman between samples of both synthetic and biogenic magnesian–calcites (Bischoff et al., 1985). Thus, Raman spectroscopy
may not be sensitive enough to detect such relatively small fluctuations of Mg concentration in calcite.
3.5. Cellular observations at micro- and ultrastructural level
In semi-thin (ca. 1 lm) sections through the ectosome outer
part of the sponge body wall, stained by toluidine blue-Borax in
ethanol 70% (Fig. 7a and b), growing spicules are associated with
formative sclerocyte cells localized in the mesohyl, sometimes almost in contact with the surface pinacoderm (Fig. 7a). Their shape,
orientation and localization in the ectosome suggest that they
probably correspond to immature giant tetractines or triactines.
At this stage of spicular growth, one sclerocyte is positioned subapically at the tip of each ray (Fig. 7a and b). The maximum width
of the ray to which they are associated varies between 4.5 and
10 lm (values obtained from six different rays covered with a
sub-apical sclerocyte). SEM examination of the choanosome inner
part of a cryofractured sponge body wall (Fig. 1b) also reveals some
sclerocytes associated with growing spicules (Fig. 7c and d). A
short, straight and very thin (less than 500 nm wide) microdiactine
probably at its initial stage of growth is lodged in an intercellular
cavity bounded by two formative sclerocytes (Fig. 7c). At a later
stage of spicular growth, two sclerocytes are positioned sub-apically, each associated to one of each tip of the microdiactine
(Fig. 7d).
The morphology of sclerocytes varies from cuboidal to flattened, more or less elongated and with some pseudopods
(Fig. 7a–d). In semi-thin sections, they display a nucleus approximately 5 lm in diameter and intracellular granules stained with
toluidine blue (Fig. 7a and b). At the ultrastructural level, they
are characterized by numerous mitochondria and a great abundance of small vesicles with homogeneous electron-translucent
C. Kopp et al. / Journal of Structural Biology 173 (2011) 99–109
105
Fig. 6. Raman microspectroscopy analysis of an apical ray from a giant tetractine of Leuconia johnstoni revealing that this spicule consists entirely of magnesian–calcite. (a)
Optical image of a transverse section of the apical ray. (b) Raman spectra were recorded from the center, the medium and the extreme periphery (respectively noted (1), (2)
and (3)) of the surface of the section. The three spectra exhibit bands characteristic of magnesian–calcite: two lattice modes at 156 cm 1 and 283 cm 1, t1 CO3 symmetric
stretching mode at 1091 cm 1 and t4 antisymmetric stretching mode at 716 cm 1. The weak broad band observed near 1010 cm 1 may be attributable to bicarbonate
(HCO3 ) ions (Bischoff et al., 1985). (c) Raman map of the section showing the correlation index of each spectrum with the reference spectrum of lattice vibrations in
crystalline calcite. A value of 1 indicates a perfect similarity of the sampled spot and the reference and any value above 0.95 an excellent similarity. (d) Raman image of the
Raman shift of the t1 CO3 symmetric stretching mode. (e) Raman image of the full width at half maximum (FWHM) of the t1 CO3 symmetric stretching mode. For (c, d), the
signal obtained on the outside of the ray is due to the basal triradiate system situated at the bottom.
granular content (Fig. 7e and inset). Sclerocytes also contain a few
electron-dense granules (Fig. 7e and inset).
A very thin organic sheath is sometimes detected enveloping
growing and mature spicules (Fig. 7a and b). After spiculogenesis
this sheath may be thickened by addition of mesohyl-derived collagenous fibrils secreted by collencytes that were detected very
close to the spicules (data not shown). Mature, cell-free spicules
are observed to be positioned and anchored into the collagenous
matrix of the mesohyl through an oriented network of collagenous
fibrils (Fig. 7f and inset) with protein complexes closely associated
to the tip of each spicule ray and to the collagenous fibrils.
4. Discussion
4.1. Hierarchical structure of calcareous sponge spicules
Microstructural organization of calcareous spicules, characterized by successive concentric layers, strongly suggests that biomineralization in calcareous sponges is a cyclical process: during
spicular growth, successive layers are secreted by sclerocytes and
accreted around the axis of actines.
At micrometric length scale, acidic etching of polished transverse and longitudinal spicule sections revealed that the giant tetractines of L. johnstoni consist of concentric layers of calcareous
material surrounding a central actine axis. Some primary bands
less than 250 nm wide were detected inside larger secondary
bands averaging 535 ± 260 nm in width. The occurrence of this
concentric banding pattern confirms previous SEM investigations
made with or without etching in spicular sections of other calcareous sponge species, like L. nivea (Jones and James, 1972; Ledger
and Jones, 1991), Sycon ciliatum (Ledger and Jones, 1991) and Pericharax heteroraphis (Sethmann and Wörheide, 2008). However, the
sizes of secondary bands (550–6660 nm wide) and primary bands
(100–900 nm wide) reported in giant triactines of L. nivea (Jones
and James, 1972; Ledger and Jones, 1991) were larger than those
we observe for giant tetractines of L. johnstoni. Banding pattern
may be specific to the species and to the spicule type, but reflects
an incremental growth process general to all calcareous sponge
spicules.
The factors responsible for this banding periodicity are certainly
intrinsic to sclerocyte activity but still need to be discovered. As
concluded by Ledger and Jones (1991) for small spicules of Sycon
sp, the number of growth layers in an actine is too high to correlate
with tidal or circadian rhythms, taking into account that one spicule takes no more than two days to fully grow. This rapid rate of
skeletal deposition was confirmed for Sycon sp curved monaxon
by in vivo fluorescent calcein-labeling studies (Ilan et al., 1996),
establishing a 12 ± 3 lm/h growth-rate and full secretion within
about 24 h, although growth rate of other spicules types varied.
The near rectilinear ridge we observed in cross-section from giant
tetractines of L. johnstoni could indicate a crystallographic continuity from one layer to another. Thus, this suggests that a new layer
may crystallize in continuity to the precedent deposit.
At the nanostructural level, combined AFM and SEM results
showed that spicules of L. johnstoni are polycrystalline and consist
of a multitude of nanograins commonly ca. 60–130 nm in diameter, intercalated with spaces of a few nanometers. Nanogranular
organization, with nanograin size ca. 10–50 nm, has also been reported in spicules of the calcareous sponge P. heteroraphis by Sethmann et al. (2006) based on AFM observations. Each nanograin was
interpreted as being composed of several crystal domains of a few
nanometers, visible with high-resolution TEM, and possibly
embedded in organic matter (Sethmann et al., 2006). This nanoorganization of calcareous sponge spicules may be responsible
for the conchoidal fracture pattern observed when they are broken
(Figs. 4c, 5f and 6a), which is different from the cleavage pattern
characteristic of abiogenic calcite. Indeed, this fracture property
106
C. Kopp et al. / Journal of Structural Biology 173 (2011) 99–109
Fig. 7. Spicule-secreting sponge sclerocytes. (a) Optical image of a semi-thin section of the sponge ectosome region stained with 1% ethanolic toluidine blue-borax showing
three growing rays (outlined with lines) of an immature giant tetractine or triactine: each ray tip is associated with a sub-apical sclerocyte. (b) Enlargement of a sclerocyte on
another spicule growing in the ectosome. (c) SEM micrograph of the cryofractured choanosome region of the sponge with a short, straight and very thin (less than 500 nm
wide) microdiactine at its initial stage of growth, lodged in an intercellular cavity bounded by two formative sclerocytes, in the ceiling of a choanocyte chamber. (d) SEM
micrograph of a microdiactine at a later stage of growth with an associated sub-apical sclerocyte. (e) TEM ultra-thin micrograph of a sclerocyte showing a great abundance of
mitochondria and small electron-translucent vesicles and a few electron-dense granules. Inset is higher magnification view of the cell ultrastructure. Holes at the bottom of
section correspond to spicule fragments fallen during sectioning. (f) TEM image of an ultra-thin section through a mature spicule positioned and anchored into the
collagenous matrix of the mesohyl through an oriented network of collagenous fibrils associated with protein complexes at the tip of each spicule ray. edg = electron-dense
granule; me = mesohyl; pc = protein complexes; pi = pinacoderm; ps = pseudopod; scl = sclerocyte; sh = organic sheath; sv = small vesicles; sp = spicule; sw = sea-water.
might be the result of the deflection of cracks propagating at the
boundaries of cluster-nanograins or crystal domains as suggested
by Sethmann et al. (2006). The nanogranular organization of calcareous sponge spicules thus confirms that nanogranular components are widely distributed among organisms producing calcium
carbonate biocrystals under biological control, suggesting that they
are a universal component of biogenic carbonates (Stolarski and
Mazur, 2005).
Interestingly, TEM observations of partially decalcified microdiactines revealed a network of intraspicular organics, delineating
small compartments of approximately the same size as nanograins
observed with AFM. Therefore, taking into account their positive
staining with Ruthenium Red, indicative of glycoproteins, we can
suppose that each nanograin may be surrounded by glycopro-
tein-rich intraspicular organic material. Aizenberg et al. (1996b)
characterized the intraspicular proteinaceous macromolecule fraction representing 0.07–0.1 wt.% of the spicules of Sycon sp, Kebira
sp and Clathrina sp, revealing a content rich in asparagine and/or
aspartic acid. Based on synchrotron X-ray diffraction analysis and
in vitro precipitation experiments, Aizenberg et al. (1995a, 1995b,
1996b) suggested that unidirectional elongation of the spicule rays
was controlled by stereochemical interactions of the growing crystals with intraspicular specialized proteins. As acknowledged in
many other biomineralization models (Weiner and Dove, 2003),
intraspicular organic components of calcareous sponge spicules
may function as mineralizing organic matrix serving as a template
for the nucleation and subsequent growth of the fundamental spicule biomineralization units.
C. Kopp et al. / Journal of Structural Biology 173 (2011) 99–109
4.2. Existence of a compositionally distinct core in the giant tetractines
of L. johnstoni
The occurrence and the nature of a differentiated core in calcareous sponge spicules have been debated for a long time (e.g. Minchin, 1909). In this work, we used high-spatial resolution
NanoSIMS analysis to show that the core of rays from giant tetractines of L. johnstoni is significantly depleted in Mg compared to the
adjacent envelope. The measured Mg/Ca ratio in the 3.5–8 lm
wide core is 70–90 mmol/mol compared to 125 to 130 mmol/
mol in the envelope. This compositional span is in agreement with
the range of values obtained for spicules of others calcareous
sponge species (Table 1). Moreover, preliminary SXM analyses
indicate that the core of rays of giant tetractines is also enriched
in S compared with the envelope. The S detected may be in the
form of sulfated polysaccharides embedded in the organic matrix
of the biomineral, as it is often the case for other biocarbonates like
the aragonitic skeleton of scleractinian corals (Cuif et al., 2003). We
did not detect any periodic variation in the Mg and S concentration
in the concentric layers, which might have been expected considering the banding pattern observed at the microstructural scale.
These results contrast with those of Jones and James (1969)
who did not detect fluctuations in the distribution of Ca, Mg, Sr
and S from the center towards the periphery of transverse and longitudinal sections of giant triactines of L. nivea. The relatively low
spatial resolution and sensitivity of the electronic micro-probe
they used likely provide an explanation for this discrepancy. Using
energy-filtering TEM on crushed triactines of P. heteroraphis, Sethmann et al. (2006) observed a heterogeneous distribution of Mg.
However, because the sample was crushed into powder and the
analysis was conducted at the nanoscale, it was not possible to correlate these Mg fluctuations with potential microstructural features like a core or a banding pattern. Our results confirm the
pioneer hypothesis of Von Ebner (1887), suggesting that the central part of rays may be compositionally distinct from the peripheral part of rays. Our results also support the hypothesis of Jones
(1970) who suggested, based on the fact that the relatively small
spicules of L. nivea and A. paulini contained slightly less Mg than
larger spicule types (Jones and Jenkins, 1970), that the material
first secreted (which we refer to as the core) was depleted in Mg
compared to the later deposits (which we refer to as the envelope).
Table 1
Compared magnesium concentration in calcareous sponge spicules.
Species
Mg/Ca (mmol/mol)
in the spicules
References
Leuconia johnstoni
70–90 in the core
and 125–130 in the
envelope
104–114
This work, measured
in giant tetractines
with the NanoSIMS
Jones and Jenkins
(1970)
Leuconia nivea
Leucandra (=Leuconia)
pumila
Clathrina coriacea
Sycon ciliatum
Grantia compressa
Amphiute paulini
Leucosolenia complicata
Leucosolenia eleanor
Leucilla (=Rhabdodermella)
nuttingi
Clathrina contorta
98
Sycon sp
Kebira uteoides
Pericharax heteroraphis
Leucetta sp
125–135
140–180
100–110
90–120
129
52–54
78
79–90
82
65
69
160
107
The existence of a compositionally distinct core may also be
suggested for spicules of other calcareous sponge species, based
on reports of central etching pits sometimes observed on ray sections, for example in giant triactines of L. nivea treated with 10%
D-tartaric acid for 30 s to 3 min (Jones and James, 1972; Ledger
and Jones, 1991). We did not see such corrosion figure in the giant
tetractines of L. johnstoni etched with 0.1% formic acid during
2 min 15 s: in fact, no distinct microstructure was observed in
the spicule center, which could be related to the size of the compositionally differentiated core, corresponding to the 2–4 first layers
around the axis of actines. As emphasized by Jones and James
(1972), the etching pattern may differ depending upon the etching
agent, its concentration and its time of application.
Minchin (1898) and Minchin and Reid (1908) observed in decalcified calcareous sponge spicules from several species (treated
with picric, nitric, acetic or hydrochloric acid), the presence of an
axial filament stained with nigrosin or indulin, suggesting an organic phase. However, Jones (1967) and then Ledger and Jones
(1991) established that this putative organic axial filament was
an artifact caused by contracted remnants of the decalcified spicule
organic sheath. In cryofractured or etched sections of spicules of L.
johnstoni examined with SEM, we did not observe any organic axial
filament. Today, the general consensus is the absence of an organic
axial filament in calcareous sponge spicules (Sethmann and
Wörheide, 2008), unlike the proteinaceous axial filament observed
in all siliceous sponge spicules (Uriz et al., 2003; Uriz, 2006).
Raman mapping results show that the giant tetractines of L.
johnstoni, as well as its smaller minute tetractines, microdiactines,
pugioles and triactines, consist entirely of magnesian–calcite.
There are no differences in terms of crystallography between the
core and the envelope of the spicules. In contrast, the spicules of
Clathrina sp (from Atlit, Israel) are reported to be composed of a
calcitic core, enveloped by a thick layer of ACC, which may be covered by a thin calcitic layer (Aizenberg et al., 1996a, 2003). In our
work, no ACC was detected with Raman microspectroscopy. It is
possible that the presence of ACC in calcareous sponge spicules
may be species specific, revealing different patterns of biomineralization among the spicule types in the Calcarea. We can also not
rule out that a potential amorphous phase may have accidentally
crystallized into calcite during the protocol of spicule preparation
or during the Raman analyses. Based on our results it is not clear
whether an unstable, transient, amorphous precursor phase of calcite might exist in calcareous sponges, as has been reported in several Metazoan groups like Echinoderms, Crustacea and Annelida
(reviewed by Weiner et al., 2009). The higher Mg content we have
detected in the spicule envelope as compared to its core could be a
signature of transient ACC during spicule formation (Loste et al.,
2003). The abundance of Mg may be regulated by a spatial difference in the composition of the intraspicular organic matrix. Wang
et al. (2009) recently reported experimental in vitro regulation of
Mg/Ca in carbonates with carboxylated acidic molecules, which
are known to exist in sponge spicules (Aizenberg et al., 1996b).
In any case, the tightly controlled spatial fluctuations we have observed in the spicule geochemical composition indicates a strong
biological control exerted by the sponge organism on its skeletal
biomineralization and can only be explained by differences in spicule-secreting sclerocyte cell types or metabolic activities, suggesting a two-step cellular growth process.
Chave (1954)
Aizenberg et al.
(1995a)
Sethmann et al. (2006)
Uriz (2006)
4.3. Two-step cellular model of spiculogenesis for the giant tetractines
of L. johnstoni
Earlier optical and electron microscopy observations have provided the basis for a cellular model of calcareous sponge spicule
formation for small monaxons, triactines and tetractines of sponge
species of the genus Leucosolenia, Clathrina and Sycon (Minchin,
108
C. Kopp et al. / Journal of Structural Biology 173 (2011) 99–109
Longitudinal section
Growth direction
Apical sclerocyte
Lateral sclerocyte
Concentric band
= Core of ray (depleted in Mg
and enriched in S)
Envelope of ray (enriched in
= Mg and depleted in S)
Fig. 8. Cellular model of spiculogenesis of the giant tetractines of Leuconia johnstoni. Spicule actines grow by successive addition of concentric layers secreted by two types of
formative sclerocytes: one apical sclerocyte ‘Founder-Cell’ depositing the core of ray which is depleted in Mg and enriched in S (axial growth), and lateral sclerocytes
‘Thickener-Cells’ depositing the envelope of ray, enriched in Mg and depleted in S (radial growth).
1909; Jones, 1970; Ledger and Jones, 1977). Two formative sclerocytes are required for the development of monaxons and for each
ray of a triactine or the basal triradiate system of a tetractine.
Therefore, the formation of triactines or of the basal triradiate system of tetractines begins within a group of three paired cells,
termed a ‘‘sextet” by Minchin (1909). The fourth ray of a tetractine
is added to the growing triradiate system after it has reached a certain size. Minchin (1909) stated that the extension in length of
small monaxons and of each ray of small triactines or the basal triradiate system of a tetractine is assured by one of the two sclerocytes, named the ‘Founder-Cell’, in the course of its migration
through the mesohyl. The extension in width is provided by the
second sclerocyte, named the ‘Thickener-Cell’.
Fluorescence studies with calcein-labeling have shown that
spicule growth rates and mechanisms are different for each spicule
type of calcareous sponges: triradiate spicules are deposited from
the center towards the rays, whereas curved monaxons elongate
unidirectionally, with separate, additional, lateral calcification
(Aizenberg et al., 1996b; Ilan et al., 1996).
We observed two formative sclerocytes associated with a microdiactine at an early stage of growth in the cryofractured choanosome
of L. johnstoni. We also observed that only one sub-apical sclerocyte
was associated per ray of a growing triactine or giant tetractine spicule in semi-thin sections of the sponge ectosome. Taking into account the huge size of rays of the L. johnstoni giant tetractines and
their concentric growth pattern, we propose that each tetractine
ray is generated by incremental growth involving more than two formative sclerocytes. A cellular model of giant tetractine formation is
proposed in Fig. 8. The spatial heterogeneity we detected in spicule
trace element composition is compatible with a spatial heterogeneity in cell type or activity. Indeed, the maximum width of immature
rays of growing triactines or giant tetractines measured in the contact zone under the sub-apical sclerocytes varies between 4.5 and
10 lm, which corresponds to the size of the compositionally differentiated core of the giant tetractines (3.5 to 10 lm). We suggest
that the depletion in Mg and enrichment in S observed in the core
of rays reflects the activity of a sub-apical sclerocyte ‘founder cell’
controlling axial growth, working in a distinct mode compared to
several lateral sclerocytes ‘thickener cells’ controlling radial growth
and accretion of the ray envelope relatively enriched in Mg and depleted in S (Fig. 8). This model is supported by the two-stage model
proposed for Sycon sp curved monaxon deposition based on fluorescence studies, with an apical cell forming a primary nucleation site
and a thickener cell spatially constraining lateral growth (Aizenberg
et al., 1996b; Ilan et al., 1996). Our results indicate that sclerocyte
cells would not only control skeletal morphology via the shape of
the space and macromolecule content of the spicule microenvironment, but that they also control trace element incorporation into
the calcareous spicule. Similar mechanisms of biological control of
calcium carbonate biomineralization have been proposed for other
phyla with calcified skeleton, such as the scleractinian corals (e.g.
Meibom et al., 2008). Future studies may involve detailed ultrastructural observations of the sclerocyte-spicule interface and dynamic
studies with stable isotope labeling of Mg trace element incorporation into the calcitic skeleton in order to provide information on
the fundamental mechanisms of the biological control of marine carbonate biomineralization.
Acknowledgments
We thank the director and staff of the Concarneau Marine Station
of the MNHN for help during field collection of sponges and for providing access to aquarium facilities. A number of colleagues are
thanked for inspiring discussions and technical help: Pr. Claude
Lévi, Karim Benzerara, Chloé Brahmi, Maciej Mazur, Sylvain Pont
(Direction des Collections du MNHN) and Gérard Mascarell (Plateforme de Microscopie Electronique du MNHN). The National NanoSIMS facility at the Muséum National d’Histoire Naturelle was
established by funds from the CNRS, Région Île de France, Ministère
délégué à l’Enseignement supérieur et à la Recherche, and the Muséum itself. J. Stolarski’s research was supported by funds from the
Direction des Collections du MNHN and from the Polish Ministry
of Science and Higher Education Project N307-015733. This work
was funded in part by the Action Transversale du Muséum ‘Biomineralizations’. The Raman micro-spectrometer at IMPMC was funded
by an ANR jeune chercheur grant (GeoCARBONS) to O. Beyssac.
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