Cell Tiss. Res.

181,553-567 (1977)

Cell and Tissue

Research
9 by Springer-Verlag 1977

Spicule Formation in the Calcareous

Sponge Sycon ciliatum
Philip W. Ledger* and W. Clifford Jones
Department of Zoology, University College of North Wales, Bangor, Gwynedd LL57 2UW, U.K.

Summary. The spicule primordium is formed in an intercellular cavity within

a group of sclerocytes. This cavity contains organic material which ensheaths
the growing spicule but does not appear to determine the nature of the mineral
morph (magnesian calcite) or the crystallographic orientation of the spicule.
The tip of each growing spicule ray is seated in a 'dense cup' in the cytoplasm
of the sclerocyte concerned. Both ends of monaxons are initially inserted each
into a dense cup. As rays elongate the sclerocyte membrane around the tip
becomes invaginated and forms a system of 'converging spaces' that possibly
indicate high secretory activity in that region. Spicule growth involves the
displacement and expansion of the organic sheath by the enlarging spicule.
Fully formed spicules which are exposed to the mesohyl become surrounded
by collagen fibrils. However, these fibrils are in no way concerned with the
process of mineral deposition and are never found within the spicule calcite.
Key words: Spicules - Sponge, Sycon ciliatum - Calcite - Biomineralization.
Introduction

The Calcarea (Bowerbank) is a class of sponges characterised by the possession

of a skeleton of calcareous spicules. Analyses of spicules from several calcareous
sponges, including Sycon, have shown that they are composed of impure magnesian
calcite, with a magnesium content ranging from 5.2-12.9 Molar ~ MgCO 3 in
the species studied (Jones and Jenkins, 1970). Each spicule can be considered as
a single crystal of impure calcite (von Ebner, 1887; Jones, 1955a). The orientation
Send offprint requests to: Dr. W.C. Jones, Department of Zoology, U.C.N.W., Bangor, Gwynedd
LL57 2UW, U.K.
Acknowledgements. The authors wish to express their appreciation of the technical assistance given
by Mr. D.A. Davies and Mrs. G.M. Walker of the electron microscope suite. This work is part of that
submitted by P.W.L. for the degree of Ph.D. in the University of Wales and was supported by a
Science Research Council (U.K.) Research Studentship.
* Present address: Laboratoire d'Histologie et Biologic Tissulaire, Universit6 Claude-Bernard,
43 boulevard du 11 Novembre 1918, 69621 Villeurbanne, France

554

P.W. Ledger and W.C. Jones

of their rays bears a precise but variable relationship to the crystallographic axes
of the mineral (Jones, 1954a, b, 1955a, 1970), which will henceforth be referred
to simply as 'calcite'. It has long been accepted that spicules are enveloped by an
organic sheath (Jones, 1955 b, 1967) but the presence of other organic material is
somewhat in dispute. Jones (1967) concluded that no visible organic material is
present in the calcite of Leucosolenia spicules and that the so-called axial filament
revealed when spicules are decalcified is undoubtedly an artefact. On the other
hand, Travis (1970) considered that a complex matrix of organic 'compartments'
and 'sub-compartments' is present in Sycon spicules of a certain size.
Spicules are formed in the mesohyl by groups of sclerocytes (the terminology
used in this paper is that of Borojevi6 et al. (1968). The most extensive account of
spicule formation in Sycon is that by Woodland (1905). Previously Minchin had
carried out similar studies on Clathrina (1898) and Leueosolenia (1908) and the
processes, which have been summarized by Jones (1970), proved to be similar
in the three genera. Briefly, each monaxon arises within a pair of sclerocytes and
each three-rayed triact is formed within a group of six sclerocytes, termed a
sextet. In the former case, one of the two cells migrates through the mesohyl, laying
down the spicule as it goes. This sclerocyte was termed the 'founder cell' by Minchin
(1908). When the founder cell reaches the choanoderm it halts, but continues
secreting, so that the distal end of the spicule passes through the other cell of the
pair and onwards, until it protrudes from the sponge into the surrounding sea
water. This other sclerocyte eventually migrates along the spicule towards the
founder cell. Because of its apparent function of laying down more calcite, it was
termed the 'thickener cell' by Minchin (1908). Woodland (1905) found that this
thickening activity was less noticeable or even absent in Sycon, but Minchin's
terminology will be used here for convenience. In the formation of a triact the
three sclerocytes of the sextet which lie against the choanoderm (the founder cells)
move out over that layer, again laying down the rays as they go. The other three
cells initially remain at the junction of the rays but eventually migrate along their
respective rays towards the founder cells (Woodland, 1905; Minchin, 1908) and
again may or may not perform some thickening activity. In Sycon the founder and
thickener cells of both monaxons and triacts leave the fully formed rays (Woodland, 1905).
In the first electron micrographs of calcareous sponge sclerocytes (Leucosolenia) Jones (1967) noted cell junctions around a spicule ray and thus determined
that sclerocytes are separate cells, not parts of a syncytium. It has since been found
(Ledger, 1975) that this condition exists even at the sextet stage and that the junctions are typical septate junctions, unlike the simple junctions that usually occur
between cells of other types in sponges.
The present paper describes ultrastructural features of the sclerocytes and
spicules and considers them in relation to the processes involved in spicule formation.
Material and Methods
Specimens of Sycon ciliatum (O. Fabricius) [= Scypha ciliata (F), Burton, 1963] collected from the
landing stage at Menai Bridge, North Wales, were fixed immediately, or after limited maintenance

Spicule Formation in Sycon ciliatum

555

(2 days) in a sea water aquarium. Pre-fixation was carried out at room temperature in 2.5~ glutaraldehyde in 80% filtered sea water adjusted to pH 7.8 with 1.0 M NaOH (1-11/2h). This was followed
by rinses in filtered sea water at pH 7.8 (3 x 5 min and 3 x 20 min). Post-fixation, at room temperature,
was in 1~o osmium tetroxide in sea water at pH 7.8 (1 11).Material was then rinsed in filtered sea water
and dehydrated in 25~, 50~, 75~ ethanol (5-10min each) and absolute ethanol (3 lots, each of
20rain). Some material was embedded in a standard araldite mixture, some in Spurr's low viscosity
medium. Thin sections were cut with a diamond knife and double stained with uranyl acetate and
lead citrate. Variations in the method were: (1) block staining of material with 2~ uranyl acetate
for 30 rain after the last rinse and before dehydration; (2) saturation of all aqueous media, from the
fixatives to the 75~ ethanol, with calcium and magnesium carbonates.

Observations
Figure 1 illustrates the distribution of cell types as seen in a low power micrograph
of Sycon. Spicules are present at several stages of growth and, because of their
various orientations, the sectioned rays present round to oval profiles. The appearance of the spicules depends u p o n the method of preparation used. When fixed
in media without added carbonates, the spicules undergo a variable amount of
dissolution and the electron dense calcite is wholly or partly replaced by the embedding resin of much lower electron density (e.g. Figs. 6 and 9). When material
is block-stained with uranyl acetate, the acidity of the stain completely decalcifies
the spicules (e.g. Figs. 4 and 8). Partial dissolution of calcite during fixation and/or
dehydration is only prevented by the use of aqueous media saturated with calcium
and magnesium carbonates. Material treated in this way is still liable to lose its
calcite, either by dislodgment during sectioning or by dissolution in the liquid of
the knife trough, or during staining of the sections. In such cases the calcite is represented by an electron lucent hole (Fig. 11) that is easily distinguished f r o m the
resin-filled space replacing calcite dissolved before embedding. Observations of
material prepared in this way confirm that the sheath intimately invests the calcite
but is not itself impregnated with mineral. Also, no sheaths are seen that did not
enclose calcite. Because of this, the sheaths in decalcified material are taken to
indicate the presence of spicules, but not necessarily their original shape, which
would probably have been altered by shrinkage and other distortion.
Sclerocytes and early stages of spicule formation are c o m m o n l y encountered
in rapidly growing parts of Sycon. Groups of cells such as the one shown in Fig. 2
are presumably sextets, not all six cells necessarily being represented in the plane
of the section. Some of the sclerocytes are usually in contact with the c h o a n o d e r m
or endopinacoderm. As has been described (Ledger, 1975), all cells of a sextet are
joined by septate junctions (arrows, Fig. 2) and are variously interdigitated.
Certain fortuitous serial sections passed through tiny spicule primordia (for
example, Fig. 3). As these sections were from decalcified material the primordia
contained no mineral. It could be argued that calcite had not been dissolved away
and that they represented a pre-calcification stage. However, in non-decalcified
preparations for optical microscopy, refringent spicules of the same size and
smaller can be seen. Thus there is justification for supposing that in Figure 3 the
space in the p r i m o r d i u m was left by dissolution of calcite and that the surrounding
organic material is the sheath. On the other hand, another p r i m o r d i u m was somewhat smaller, less easily divisible into sheath and spicule, and m a y well have been

556

P.W. Ledger and W.C. Jones

$W

;scl

at

iom
~0 :!i::ii::i:
0~
J\
rc

Fig. 1. A diagram, based on tracings of low power micrographs, representing a longitudinal section
through the wall of S. r
in the region of chamber formation. Arrows indicate the direction of
flow of water through a porocyte (po), through the radial chamber (rc), into the atrium (at) and
towards the osculum. Spicules are represented by shaded areas; scl, two groups of sclerocytes,
probably sextets; spl , a developing monaxon within its founder (fo) and thickener (th) cells; sp2 , a ray
of an older spicule surrounded by only a thin layer of cytoplasm; sP3 , a fully formed spicule exposed
to the mesohyl (m); c, choanocyte collars; ch, choanoderm; en, endopinacoderm; ex, exopinacoderm;
fg, choanocyte flagella; sw, sea water, x 700

Fig. 2. A group of sclerocytes (sc/), probably a sextet. Arrows indicate the position of the septate
junctions. The group of cells is in contact with the choanoderm (ch) and encloses an intercellular
cavity (ic). m, Mesohyl; n, nucleus; sw, sea water, x 5890
Fig. 3. A triact primordium represented by its sheath (sh) surrounding a space left by dissolution of
the calcite (sp). The intercellular space containing the primordium is continuous with the larger
intercellular cavity (ic). Adjacent to the primordium, within the sclerocytes, is dense material (d) of
the dense cups. In the lower part of the micrograph numerous interdigitations of the sclerocytes result
in a highly convoluted septate junction (sj). 26,600

Fig. 4. A developing triact seen in section with the tip of a ray surrounded by a well developed system
of converging spaces (co) in the founder cell (Fo). Arrows indicate the positions of the septate junctions.
ch, Choanoderm; g, Golgi apparatus; m, mesohyl; n, nucleus; sh, sheath; sp, space left by dissolved
calcite; sw, sea water, x 5605

558

P.W. Ledger and W.C. Jones

an earlier stage in which little or no calcite had been deposited. Immediately adjacent to the primordia, within the sclerocytes, are regions of finely granular
cytoplasm. Although this dense material is often irregularly disposed in sclerocytes of early stages, later it is invariably present as a cup-shaped body situated
on the tip of each and every growing ray (for example, Figs. 6 and 9). It will be
referred to as the 'dense cup'. Optimal sections have indicated that even at this
intimate point of contact between spicule and sclerocyte the unit membrane of the
latter is continuous. The section shown in Figure 3, which was in the plane approximately perpendicular to the choanoderm, displays only two sclerocytes and two
dense cups adjacent to the primordium. Sections parallel to the choanoderm and
passing through the three rays of a primordium have not been obtained. However,
the interdigitating nature of the sclerocytes and the invariable association of a
dense cup with the tip of a developing ray makes it probable that in a sextet there
are three dense cups, each in a different founder cell and each adjacent to one of
the three presumptive rays of the primordium.
In their early stages of growth, up to about 5.0 txm, the rays of triacts are little
more than the corners of a triangular lump of calcite. An oblique section through
such a stage is shown in Fig. 8, in which the migration of the founder cell has
only just begun and the sclerocytes still form a reasonably compact clump of cells.
Adjacent to the dense cup in the founder cell are several invaginations of the membrane lining the cavity containing the spicule. As the growth of a spicule proceeds
(Fig. 4) these invaginations come to form a complex system of spaces that converge
onto the ray tips. Such 'converging spaces' are found around the growing tips of
rays of all types of spicules (Figs. 5, 6 and 9).
Sections of tiny monaxon primordia have not been obtained. However, pairs
of interdigitated cells joined by a septate junction and enclosing an intercellular
cavity have occasionally been seen, which may be sclerocytes paired prior to the
formation of a monaxon. The presence of a dense cup at the proximal end of
monaxons has already been noted (Fig. 6). It is also found that, before its protrusion, the distal tip of a monaxon is also seated in a dense cup in the thickener
cell and is surrounded by converging spaces. This suggests that dense cups are
associated with both extremities of a monaxon primordium and that growth
occurs at both ends, in the initial stages at least.
Spicule rays grow to a length of more than 100 ~tm and thus many of the spicules
randomly presented in a section give little information about the rapidly growing
tip region. Most of the cytoplasm of sclerocytes on a long ray is accumulated around
the cell nuclei (Fig. 7) and much of the spicule is invested by a cytoplasmic sheet
Fig. 5. A transverse section through the tip of an apical ray of a tetract. Micrographs such as this
demonstrate that the converging spaces (co) are irregular spaces rather than cylindrical tubules.
sh, Sheath; sp, dissolved spicule, x 23,750
Fig. 6. The end of a monaxon in its founder sclerocyte (Fo). At the tip the sheath (sh) outlines the
space left by dissolution of the calcite (sp). Further back towards the thickener cell (Th) the sheath
has been sectioned more tangentially. Note the dense cup (d) and converging spaces (co) around the
spicule tip and the contact between the founder cell and the choanocyte (ch). Arrows indicate the
positions of the septate junctions, g, Golgi apparatus; rn, mesohyl; mi, mitochondria; n, nucleus;
po, porocyte; sw, sea water, x 13,300

Fig. 7. A typical section through a large spicule ray (sp, sh). ch, Choanoderm; m, mesohyl; n, nucleus;

sw, sea water. 3705

Fig. 8. A triact, present as the sheath (sh) and space left by the decalcified spicule (sp). A founder
cell (Fo) contains a dense cup (d) and the first signs of the converging spaces (co). Typical features
include the contact between the founder cell and the choanoderm (ch), the numerous thin extensions
of the sclerocytes and the cytoplasmic organelles such as phagosomes (p), mitochondria (mz) and the
nucleus (n). Arrows indicate the positions of the septate junctions, f, Collagen fibrils; m, mesohyl;
sw, sea water. 4940

560

P.W. Ledger and W.C. Jones

which is as little as 50 nm thick in places. Thus, according to the plane of the section,
the cytoplasm around a ray can have an extremely variable shape. Migration of
sclerocytes along the rays gradually exposes the latter more and more to the
mesohyl, whereupon they acquire a thicker 'sheath' which is an accumulation of
the thin fibrils of the mesohyl (Jones, 1967; Ledger, 1974).
In this description of spicule formation the thin sheath has been taken as
indicating the extent of the spicule between sclerocytes. The section in Fig. 10,
one of a series through a dense cup, is presented to confirm that this is true even
to the extent that the mineral extends into the very tip of the dense cup. Fig. 10 is
of an oblique section just short of a monaxon tip and is from material in which no
decalcification occurred during fixation. Mineral was lost from the thin section
during staining, as explained above. No gap is present between the space previously occupied by the mineral and the surrounding cytoplasm, consisting of the
dense cup. Likewise no gap occurs in the section through the extreme tip of the
spicule (not shown here). The next section in the series, also not shown, was beyond
the spicule and no empty space was present in the dense cup.
Sclerocytes have a 4 ~tm nucleus which contains a single nucleolus and is
usually oval or pear shaped in longitudinal section, although it may be deformed
by the pressure of adjacent cells or spicules. The nuclear membrane is studded
with ribosomes and polyribosomes and is perforated by nuclear pores of about
50 nm in diameter. A centriole is present at the apex of the nucleus and in sextets
this may appear to point inwards towards the primordium. The Golgi apparatus
is found at this same end of the nucleus and consists of a stack of 5-8 cisternae
applied to the nucleus adjacent to the centriole and sometimes partially wrapped
collar-like around the tapering end of the nucleus. A rough endoplasmic reticulum
is present, usually sparingly distributed throughout the cells but occasionally
concentrated into approximately parallel arrays of cisternae in certain parts of
the cells. Profiles of mitochondria are round to oval and about 1.0 Ixm in their
longest dimension. Phagosomes and/or lysosomes are usually present, but multivesicular bodies and membrane whorls are less commonly encountered. The above
organelles are also found in the other common cell types, namely the pinacocytes
and the choanocytes. All the cell types also possess various smooth-surfaced
vesicles. However, in some sclerocytes these structures are abundant and represent,

Fig. 9. A longitudinal section through the tip of a large triact ray (sp) and its founder sclerocyte (Fo)
which is in contact with the choanoderm (ch). Thin collagen fibrils (]) are present in the mesohyl (m).
co, Converging spaces; d, dense cup; sh, sheath, x 17,100
Fig. 10. One section of a series through the tip of a monaxon. The material was not decalcified during
fixation (see text), d, Dense cup; m, mesohyl; sp, space left by removal of calcite during staining.
38,950
Fig. 11. A sclerocyte containing m a n y smooth-surfaced vesicles (sv) and/or tubules (st). Other organelles present are the nucleus (n), the Golgi apparatus (g), mitochondria (mi), rough endoplasmic
reticulum (rer) and a p h a g o s o m e (p). The material was not decalcified during fixation and thus the
sheath (sh) is closely applied to the electron-lucent space (sp) left when the mineral was lost during
sectioning or staining, x 15,675

Fig. 12. A typical secretory product of monaxon thickener cells. The filamentous material is present
in the Golgi cisternae (gc and single arrow) and in nearby vesicles (double arrow); n, nucleus, x 37,050
Fig. 13. Material in large vesicles (v) clustered around and apparently releasing their contents onto a
monaxon (sp); m, mesohy]. 24,700

562

P.W. Ledger and W.C. Jones

at least partly, a system of tubules rather than isolated vesicles (Fig. 11). Parts of
these tubules bear a coat on the cytoplasmic side of their membranes. This coat
is the same as that of the coated vesicles commonly seen within the cytoplasm of
sclerocytes, or forming invagination figures on the membrane of the spicule cavity.
Less frequently they are present on the sclerocyte membrane in contact with the
mesohyl, or on sections of junctional membrane, or attached to the edges of Golgi
cisternae. The Golgi apparatus of monaxon thickener cells produces fibrillar
material that can be seen in the periphery of its cisternae, in adjacent vesicles (Fig.
12) and in much larger vesicles that appear to release their contents around the
spicule (Fig. 13). The latter vesicles are found at the end of the thickener cells
where the monaxon is being exposed to the mesohyl. Bundles of microfilaments
are also seen in the cytoplasm of sclerocytes. They are most extensive in sclerocytes
around the older (wider) parts of spicule rays.
Although a detailed presentation of spicule structure is beyond the scope of
this present paper, decalcified spicules are present in most of the micrographs
and so it is fitting that their appearance be noted. They do not appear to contain
an ordered organic matrix, even when viewed at magnifications higher than those
shown here. The spaces left when spicules are decalcified during fixation are
usually quite empty (Figs. 5, 6 and 9). However, in material which has undergone
block staining (and thus decalcification) with uranyl acetate the spaces usually
contain variously sectioned strands of material (Figs. 4, 7 and 8) which will be
discussed in a later publication. There is good evidence that these are artefacts.

Discussion

Calcareous sponge spicules are extracellular structures, both at their initiation

and during their subsequent growth. They are formed in an intercellular cavity
bounded by a number of sclerocytes. The relationship between a triact primordium
and its surrounding sclerocytes is summarized in Fig. 14. In this the arrangement
of the septate junctions is simplified, but their suitability as barriers to isolate the
microenvironment containing the crystallization liquor is apparent (Ledger, 1975).
When monaxons are protruded through the pinacoderm, or parts of a ray are
exposed to the mesohyl, the spicules lose their complete cellular envelope. However, because the remaining sclerocytes ensheath the spicule for some length of
the ray, it is conceivable that the growing tip remains effectively isolated from the
mesohyl or sea water.
The dense cup is present at all stages of spicule growth. Its function is unknown
but it could act as a mechanical link between spicule and sclerocytes, maintaining
the correct relative position between the two. Alternatively it may be a site of
active or specialized secretion, this being suggested by the close contact, almost
coalescence, of the sheath with the rim of the dense cup (Figs. 6 and 9). The converging spaces are almost certainly sites of high secretory activity. Their importance
could be in the way they increase the area of secretory membrane. Possibly they
arise from the localized coalescence of secretory vacuoles with the membrane
of the spicule cavity. The homologous structure of the founder cells of monaxons
and triacts is not surprising. However, it is interesting to note that monaxon

Spicule Formation in Sycon ciliatum

563

Fig. 14. A diagram summarizing the

relationship betweena triact primordium
and its surrounding sclerocytes.Contact
with the choanoderm has not been indicated
because it has not been confirmedthat such
contact is restrictedto the founder
sclerocytesat such an early stage.
ce, Centriole; d, dense cup; g, Golgi
apparatus; ic, intercellularcavity;
m, mesohyl;n, nucleus;pr, primordium;
sj, septatejunction

thickener cells also possess a dense cup and converging spaces. This similarity to
the founder cells suggests that calcite secretion occurs at both ends of the monaxon
during the initial stages of growth. If so, the S y c o n monaxon would be a diact
rather than a monact, one of the two rays being rudimentary. There is no evidence
that the three rays of triacts arise independently as non-birefringent rodlets which
subsequently become fused together. This was suggested by Minchin (1898) and
Woodland (1905), but their observations were probably made on corroded preparations, as Jones (1970) has already indicated.
In addition to any secretory activity o f the membrane of the spicule cavity,
various cytoplasmic organelles are also probably involved in aspects of spicule
secretion. Virtually all of the organelles present in sclerocytes have been implicated
in relevant transporting, secreting or sequestering activities in the cells of other
organisms. However, because the sclerocytes (particularly the founder cells)
simultaneously secrete components of the mineral phase, the organic sheath and
perhaps even material of the mesohyl, it would be futile to attribute specific functions to particular organelles on the basis of the present ultrastructural evidence
alone. Perhaps the only exception to this is the activity of the Golgi apparatus
of monaxon thickener cells. The material secreted by this is apparently predominantly organic, and may form a coating on the spicule which persists when the
spicule is exposed to sea water or the mesohyl. However, a mineral-depositing
('thickening') function for these vesicles cannot be ruled out.
There is no evidence that precipitated calcium salts are stored or transported
by sclerocytes or any other cell types. Also, it is inconceivable that the sclerocytes
could contain enough calcium ions in solution to provide for the amount of
calcium contained in a fully-formed spicule. It thus appears that calcium ions
destined for spicule growth are continuously taken up from the mesohyl by the
sclerocytes. Presumably the ultimate source of supply is the surrounding sea
water. In this connexion Jones (1971) and Ledger (1976) have found that sponge
juveniles can form spicules in artificial sea water mixtures and that the amount of
spicule formation is related to the calcium ion concentration of the media.
The apparent lack of an ordered intraspicular organic matrix is consistent
with t h e observations of Jones (1967). These and more extensive observations
(Ledger, 1976; to be published) do not support the description of spicule structure
given by Travis (1970). It should be stressed particularly that although collagenous
fibrils of the mesohyl come to surround fully formed spicules, they in no way participate in the formation of the spicules and do not become calcified themselves.

564

P.W. Ledgerand W.C. Jones

The strands of material observed in the space left by spicules decalcified by uranyl
acetate could be interpreted as an organic axial filament. However, it is our
opinion that this material is art&actual, probably arising from the contraction of
material of the sheath, and that an organic axial filament is not present in calcareous
sponge spicules. Finally, while it is conceivable that soluble, non-organized intracrystalline organic material may be present within the calcite, this has not been
revealed by the ultrastructural techniques used here.
The visible organic material associated with spicule calcite is thus restricted
to the sheath. According to current theory one would expect this 'organic matrix' to be at least partly responsible for determining or controlling aspects of the
form and/or crystallographic nature of the mineral phase. To what extent can such
organic-inorganic interaction be identified in the spicules of Sycon? In a growing
ray calcite deposition occurs on, and is in crystalline continuity with, pre-existing
calcite. In no way does the sheath act as an epitactic membrane. To consider that
calcite nuclei originate in or on the sheath and grow in towards the spicule is
contrary to all observations. Also, although examination of partially decalcified
material might suggest otherwise, the mineral penetrates to the very limits of the
available space within the sheath. Thus one cannot suppose that an organic
'compartment' or 'mould' is formed in advance of the calcite. One is left with the
situation in which it is the advancing surface of the calcite itself that displaces
the sheath. Thus inorganic ions must diffuse through the sheath, which is progressively displaced by the incorporation of the ions on the crystal lattice. The
occurrence of this process is easier to accept when one considers the crystallographic technique of growing spicules in gels. In such systems the faces of the
growing crystals are supplied by ions diffusing through the supporting gel. As
the crystal grows it either incorporates or displaces the gel, depending on the particular nature of the two phases (Banks et al., 1973; Henisch, 1970). Since the
sheath is not incorporated within the growing spicule, the spicule must displace
the sheath, which here represents the gel phase. A possible objection to this
comparison is that the sheath is too thin to be regarded as a gel. However, the
important factors are only the interface between the two phases and the ability
of one to displace the other. Although this may explain how the spicule grows
despite its enveloping sheath, it is clear that numerous processes intervene to subordinate the purely physical growth of the calcite to the requirements of the sponge,
producing a spicule with a smoothly rounded surface rather than a crystal with
its characteristic faces. Although these morphogenetic factors cannot be precisely
determined at this stage the following features may be worthy of note. First,
whereas a crystal growing in a gel receives its constituent ions from all directions,
a spicule ray is probably supplied mostly at the tip. This, together with the migration of the sclerocytes, could result in the tendency for the rays to grow in a linear
fashion. Secondly, although the growing spicule displaces the sheath, one can
envisage that at some stage the sheath could come to halt its growth. Thus, if the
sheath were progressively stretched, its tension would increase; when the force it
exerted on the spicule became equal to the 'crystallization pressure' (KhaimovMal'kov, 1958) of the calcite, no further mineral deposition would occur because
the sheath could not be displaced to accommodate it. This could result in the rounded section of the rays. Certainly the sheath does appear to be under tension because

Spicule Formation in Sycon ciliatum

565

it contracts greatly when a spicule is decalcified by certain reagents (Jones, 1955b;

Ledger, 1976).
Particular problems which must be overcome during the initiation of spicule
growth concern the determination of the mineral morph and the orientation of its
crystallographic axes relative to the spicule rays. It has been seen that organic
material is present at the earliest stages of mineral deposition and may even precede
them. However, we do not consider that this material directly determines the
nature of the mineral morph by some epitactic mechanism because the material
is unordered, has no particular orientation, and surrounds the mineral from the
very start rather than becoming overgrown by it. If an organic template is absent
then another morph-determining factor must be postulated. In accordance with
general statements made by Lippmann (1973, p. 133) it seems probable that the
specific ionic composition of the crystallization liquor could be of paramount
importance in encouraging the precipitation of calcite and preventing the accidental
formation and growth of unwanted morphs, such as aragonite or vaterite. From
the work of Glover and Sippel (1967) one can see that in order to obtain magnesian
calcites with magnesium contents in the range found in sponge spicules (5.2 to
12.9 Molar % MgCO3; Jones and Jenkins, 1970) the crystallization liquor must
have a Mg/Ca ratio as high as that of sea water, about 5.5. However, the magnesium
content of such solutions usually inhibits the formation of calcite and favours the
appearance of aragonite (Simkiss, 1964; Bischoff, 1968; Lippmann, 1973) or unstable magnesian calcites (Glover and Sippel, 1967). Nevertheless, under certain
conditions the formation of stable magnesian calcites can be favoured (Towe and
Malone, 1970; Berner, 1975), particularly by the inclusion of soluble organic
compounds such as sodium pyruvate, citrate or succinate in the crystallization
media (Kitano et al., 1969; Kitano and Hood, 1965; Kitano and Kanamori,
1966). Thus the nature of the mineral morph could be determined by the composition of the secreted crystallization liquor. However, this is not to say that the
organic material plays no part in the initiation of spicule formation. Ions secreted
into the intercellular cavity presumably become dispersed in the whole cavity,
whereas spicules arise only in the vicinity of, and between, the dense cups. It is
known that even stereochemically mis-matched material can have considerable
seeding ability and that heterogeneous nucleation occurs at lower degrees of
supersaturation than homogeneous nucleation (Henisch, 1970). Thus the nonoriented, stereochemically unspecific organic material could induce nucleation
within itself, while the composition of the liquor would allow only the required
magnesian calcite to form. In other words, the organic material could determine
the site of spicule deposition (i.e. between the dense cups). It is also possible that
the sheath has Ca-Mg secretory ability, but this is unlikely in view of its ultrastructure, lacking a unit membrane and consisting of a microfibrillar organization.
Fully formed spicules have a triradial or bilateral symmetry and their form
bears a definite but variable relationship to the crystalline axes of the calcite.
Moreover, both the form and the crystallographic orientation of a spicule vary
according to the position in the sponge in which it is formed (Jones, 1954a, b,
1955 a, 1970). However, a calcite crystal deposited by the mechanism proposed
above would be randomly oriented relative to the sclerocytes and the sponge. It
is thus clear that apart from the secretory mechanisms there must also be a mecha-

566

P.W. Ledger and W.C. Jones

nism that determines the orientation of the crystalline axes. Of the mechanisms
that are possibly involved, the case for an oriented organic matrix has been discounted above and the relevance of certain uninvestigated phenomena such as
the bioelectric fields of the sclerocytes and the whole sponge cannot with present
knowledge be evaluated. The general relationship between spicule form and the
crystalline axes of the spicule calcite to some extent suggests that crystallographic
factors are, in fact, acting as crystallomorphic forces. Applying this to the primordium one can assume that the specific composition of the crystallization
liquor encourages the formation of a particular shape of crystal, as has been found
in inorganic systems (Kirov et al., 1972; Folk, 1974). The fitting of this shape to
the space available between the dense cups could then result in orientation of the
crystalline axes relative to the sclerocytes. Subsequent preferential growth of
certain surfaces might then play a part in determining spicule form. However,
as Jones (1970) has pointed out, it is difficult to envisage a simple intervention
of crystallomorphic forces because of the continuous variation of both the planar
angle (within limits) and the directions of the rays within their planes when spicules
are compared one with another. There would also be the problem of the orientation
of the space into which the calcite rudiment would have to fit (Jones, 1967), as
well as the separate problem of the mechanism of orientation of the sclerocyte
pairs, trios or sextets in relation to the morphological axes of the sponge.
To whatever extent crystallomorphic influences may be present it is clear that
the final form of a spicule is the resultant between the inorganic forces of crystallization and the subordination of those forces by the organism. In so far as the ultimate site of this interaction must be at the surface of the calcite, it is hoped that
the ultrastructural relationship between sclerocyte, sheath and spicule may be useful in the future development of concepts and hypotheses concerning the formation of calcareous sponge spicules and other, similarly organized skeletal structures.
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Accepted April 3, 1977