Crystal growth of calcium carbonate in the invertebrates

Prog. Crystal Growth Charact. 1981, Vol. 4, pp. 98 - 147. © Pergamon Press Ltd. Printed in Great Britain

0146 - 3535/81/0601 - 0098005.00/0

CRYSTAL GROWTH OF CALCIUM CARBONATE IN THE INVERTEBRATES Nodmitsu Watabe Director Bectron Microscopy Center, and Profimmr of Biology, University of South Carolina, Columbia, S.C. 29208, USA

(Submitted 5th February 1981)

i.

INTRODUCTION

Mineral formation is widely observed in living systems and twenty four inorganic compounds have been found to date to be present in various organisms (Lowenstam, 1974; Lowenstam and Margulis, 1980). Of these, calcium carbonate is the most conxnon and occurs as calcite and/or aragonite, and also to a lesser extent as amorphous CaC03, vaterite, or monohydrocalcite (Watabe, 1974; Watabe and Dunkelberger, 1979; Lowenstam and Margulis, 1980). The size, habit, and the state of aggregate of polymorphic species of calcium carbonate which build calcareous structures vary by the organisms; however, each organism normally maintains specific characteristics of these crystalline patterns. In the following sections, we shall examine the crystalline characteristics of calcareous structures, the sites of their formation, nucleation and growth of crystals, the organic matrix and other factors controlling the crystalline patterns. Discussions will be limited to those structures in the invertebrate animals in which the majority of the CaCO 3 crystals are concentrated. 2. 2.1.

CRYSTALLINE CHARACTERISTICS OF CALCAREOUS STRUCTURES

Structures Made of Sin~le Crystal s

It is not common to find calcareous structures entirely made of single crystals. A few examples are the adult skeletons and larval spicules of echinoderms, and the spicules of calcareous sponges. Interestingly, all of these crystals are high magnesium calcite. The echinoderm skeletons are test plates, spines, jaw apparatus, and ossicles, of which the first two are made of single crystals (West, 1937; Raup, 1966; Currey and Nichols, 1967; Donnay and Pawson, 1969; Nissen, 1969). An exception seems to be the cortex of primary spines of cidarid which is made of polycrystalline aggregate of calcite (M~rkel and others, 1971). Towe (1967) suggested that the inner portion of the test plate of Strongylocentrotus droebachiensis is a single crystal but the exterior is a polycrystalline aggregate with preferred orientation. The unit cell dimensions of the calcite determined in the coronal plate of Stron~ylocentrotus droebachiensis are: a = 4.932 + 0.001 ~; c = 16.773 + 0.003 (Donnay and Pawson, 1969). The dimensions are somewhat smaller than tho~e of pure calcite due to the presence of appreciable amounts of magnesium, in this case being 99

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N. Watabe

Fig. I.

A portion of a spine of an adult sea urchin Arbacia punctulata, showing a fenestrated structure. The spine is made of a single crystal of calcite. (Watabe). x 300

13.5 at wt%. The magnesium content varies by the family and perhaps by the genus, and by the environmental temperature (see section 5), but ranges roughly from 5.5 to 18.5 wt % as MgCO 3 (Weber and others, 1969). The test plates and spines indicate complex reticular or fenestrated structures (Fig. I). The central region of the solid spines such as in Strongylocentr0tus or Arbacia is a lattice of anastomosing crystal and is continuous to an outer zone of radial wedges which thickened towards the periphery of the spine (Donnay and Pawson, 1969; Nissen, 1969; Weber and others, 1969; Heatfield, 1971; Davis and others, 1972). In the hollow spines of diadematids the central region is perforated with or without inner meshwork (Mischor, 1975). Spines are elongated parallel to the c-axis, but the a-axis is oriented in many different directions (see Donnay and Pawson, 1969). In the test plate, the c-axis is oriented perpendicularly or tangentially to the plate surface (West, 1937; Garrido and Blanco, 1947; Donnay and Pawson, 1969; Raup, 1962; Nissen, 1963, 1969). In some species the c-axis is inclined to the plate surface and the angle varies by the plate (Raup, 1962; Nissen, 1969). The direction of orientation of the c-axis in relation to the morphology of the plate varies by the species, e.g. it is along the row of perforations of the plates in one species, and along the line connecting the mouth and the plate in the other (Nissen, 1969; Donnay and Pawson, 1969). According to Weber and others, (1969) the strength to weight ratio (strength is measured in ksi, i.e. IOOO pound force per square inch; weight is expressed by bulk specific gravity) exceeds that of brick and concrete. Apparently the fenestrated structure fulfills the strength and volumetric requirements of

Crystal Growth of Calcium Carbonate in the Invertebrates

i01

skeleton with a minimum amount of material. In sea urchin larvae, a pair of calcareous granules are formed in the pluteus stage, from which a pair of bilaterally symmetric skeletal spicules develops. The main axis of the left and right spicules make an angle of about 45O(Fig. 2a). Each spicule is a triradiate

Fig. 2a.

A pair of spicules of the sea urchin Arbacia punctulata fused with an angle of 45 °. (Okazaki and Inoue, 1976).

single crystal of calcite extending three radii along the(degative) directions of the three a-axes, and later changing its direction of elongation to parallel to the c-axis (Fig. 2b) (Okazaki and Inoue, 1976; Okazaki and others, 1980). With the C Post-oralrod

~

(~a3)#

Antero-hteral~d

C' Fig. 2b.

Schematic diagram showing the crystallographic orientation of a larval sea urchin spicule. If the spicule had been carved out of a single crystal of calcite, it would be oriented as shown here. C-C':optic axis of the calcite rhomb; c-c': optic axis of the spicule. (Okazaki and Inoue, 1976)

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advanced development of the larvae the spicules further develop into skeletal rods, the form of which is characteristic of each species and may be simple, thorny, fenestered, or branched, etc. (Hyman, 1955). The ratio of Mg2+/Ca 2+ + ME2 + in the larval spines is about 5/100 (Okazaki and Inoue, 1976). The c a l c i t e s p i c u l e s o f c a l c a r e o u s sponges a r e up t o a few hundred ~m l o n g , and c o n t a i n 5.2 - 12.9 m o l a r % MgCO3 (7 s p e c i e s examined by J o n e s and J e n k i n s , 1970). In L e u c o n i a n i v e a and A m p h i u t e ~ , t h e Mg c o n t e n t i n c r e a s e d w i t h t h e i n c r e a s e in the s p i c u i " e " ~ z e . Minor c o n g t f t u t e n t a r e Sr ( 0 . 2 0 - 0 . 2 2 % ) , Na ( 0 . 4 9 - 0 . 6 6 % ) , and SO4 ( 0 . 8 2 - 1 . 1 1 % ) , and t r a c e amounts o f A1, Mn, Ba, and Li were a l s o r e p o r t e d ( J o n e s and J e n k i n s , 1970). The form o f s p i c u l e s i s monoaxon, t r i a c t ( t r i r a d i a t e ) , or t e t r a c t , and t h e o r i e n t a t i o n o f t h e r a y s b e a r s a p r e c i s e b u t v a r i a b l e r e l a t i o n s h i p to t h e c r y s t a l l o g r a p h i c axes o f t h e m i n e r a l ( J o n e s , 1955a, b, 1970).

Fractured surface of spicules sometimes show concentric layers with the thickness of less than I ~m. These do not represent daily growth rings (Jones and James, 1972), because it requires only a few days for the spicule to complete its growth ( J o n e s , 1978a). A c c o r d i n g t o J o n e s (1978a), monoaxons a r e f i r s t formed i n t h e j u v e n i l e s o f Sycon c i l i a t u m a t t h e r a t e o f 2 . 8 / h r on an a v e r a g e . When t h e triradiate f o r m a t i o n commences, t h i s r a t e d e c r e a s e s t o about 1 / h r . The triradiate s p i c u l e s a r e formed a t t h e r a t e o f about 0 . 6 5 / h r . The growth r a t e o f length of monoaxons in an artificial sea water at 15 ° C was 8.85 ~m/hr initially, which declined progressively with time. In contrast, the growth of the longest unpaired rays of triradiate followed a sigmoid curve, reaching a maximum of 5.5 ~m/hr. In Leucosolenia, the rate for monoaxons and triradiates was 3.78 ~m/hr. and 1.O-2.25 um/hr, respectively. The presence of organic matrix in the echinoderm skeletons and calcareous sponge spicules has been controversial (see Wilbur, 1976; Jones, 1978b). These are the only calcareous structures in biological systems in which organic matrix have not been positively identified. The hinge ligament of bivalve molluscs is composed of pseudo-hexagonal long needles of aragonite about a few hundred K in diameter and IOO0 X in length, embedded in an organic matrix composed predominantly of protein (Bevelander and Nakahara, 1967; Marsh and others, 1978; Marsh and Sass, 1980). The crystals are elongated along the c-axes and oriented perpendicular to the growing margin of the ligament. Marsh and Sass (1980) showed that the crystals in Mya arenaria and Spisula solidissima are twinned on (IIO). Thus, contrary to the observation by Bevelander and Nakahara (1969), the needles are not single crystals. 2.2.

Structures Made of Polycrystalline Aggregates

Skeletons composed of polycrystalline aggregates are numerous. Morphologically they may be grouped into the following categories; I) small spherules and spicules; 2) spherulitic aggregates and related structures; 3) vertical aggregates; 4) horizontally layered aggregates; 5) other types of aggregates. (This grouping is used here for the convenience of discussion and it is not intended to propose a new system of classification of calcified structures.) Because of the large number, it is not possible to list all of these structures, and only a few examples will be mentioned here. 2.2.1 S p h e r u l e s and s p i c u l e s . Many i n v e r t e b r a t e s c o n t a i n numerous c a l c a r e o u s s p h e r u l e s and s p i c u l e s i n t h e i r s o f t t i s s u e s (Watabe and o t h e r s , 1976; S i m k i s s , 1976; S i m i n i a and o t h e r s , 1977). S p h e r u l e s a r e u s u a l l y s m a l l , a few m i c r o m e t e r t o a few hundreds m i c r o m e t e r i n t h e l o n g e s t d i a m e t e r , s p h e r i c a l t o o v a l and o f t e n

Crystal Growth of Calcium Carbonate in the Invertebrates several individuals fuse together to form irregular aggregates

(Fig. 3).

103 Many

Fig. 3. Vaterite spherules in the albumin gland of the freshwater gastropod Pomacea paludosa. ~Meenakshi, Blackwelder,i and Watabe, 1974). x 1800 of these spherules are composed of concentric layers of organic matrix and crystals deposited alternatively, and those such as being present in the mantle and foot connective tissues of the freshwater gastropod Pomacea paludosa show radial structure also (Watabe and others, 1976). Each crystalline layer appdars to be an aggregate of small crystals probably with preferred orientation. Man~ of these spherules contain ions such as Mg 2+, PO43- other than Ca 2+ and C03Z-. They are amorphous as well as calcite, or aragonite (see Wata~e and others, 1976; Simkiss~ 1976, 1977; Simnia, 1977). For example, the spherules in the mantle and foot of Pomacea paludosa are amorphous or sometimes~aterl~e; those in the female reproductive tissue of Pomacea paludosa are vaterite (Meenakshl and others, 1974); and those in the calciferous gland of the earthworm family Lumbricidae are calcite or amorphous CaCO 3 (Robertson, 1939). The spherules in the amphipod crustacean Orchestia are amorphous or not highly crystalline calcite~ but in Niphargus they are rhombohedral calcite (Graf, 1969). Aragonite spherules are found in the special sac in the gastropod (Meenakshi and Watabe, unpublished). Not much information is available on the orientation of crystallographic axes in these spherules. Calcareous spicules are present in the organisms such as turbellarians (Platyhelminthes), alcyonarians (Cnidaria) and molluscs. Within the mantle and lophophor of didemnids (compound ascidlans), and articulate brachiopodlssuch as Terebratulina etc. are usually platy, oval, rod, or paddle-shaped, many with various surface modifications and ornamentations. Spicules of the turbellarians Florianella bipolaris and Bertillella sp. and some aculiferan molluscs, such as

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caudofoveates and solenogastres are aragonite (Rieger and Sterrer, 1975a,b). Other organisms, e.g. the turbellaria, Acanthomsscrostomum spiculiferum; the a c o c h l i d i a c e a n m o l l u s c s , H e d y l o p s i s s u e c i c a and A r C h i d o r i s t u b e r c u l a t a a r e known to contain CaCO 3 spicules but the m i n e r a ~ y has not been determined (Rieger and Sterrer, 1975a, b). The didemnid ascidians have aragonite (see Kniprath and Lafargue, 1980; Lafargue and Kniprath, 1978) or vaterite spicules (Lowenstam and Abbott, 1975). Many alcyonarian coral spicules are made of high magnesium calcite with 5-12 wt% MECO 3 (VinoEradov , 1953; Fox and others, 1969). The spicules of the pennatulid Renilla reniformis (sea pansy) are 180 ~m, and 25 ~m in average length and ~ r , respectively. They are trilobed in cross section and characterized by the presence of grooves running parallel or obliquely to the morphological long axis of the spicule (Dunkelberger and Watabe, 1974). The spicule is composed of needles of calcite, about 0.3 ~m in diameter, which are in side-to-side contact and elongated with the c-axes parallel to the direction of the groove. In cross sections, the needles are seen to be arranged in concentric layers which indicates that the spicules are molti-layered structures, each layer being an aggregate of needles with preferred orientation. Organic matrix materials are present in between each layer and crystals as well as within the crystals (Dunkelberger and Watabe, 1974). The alcyonacean and sorgonacean spicules are usually up to a few hundred ~m long, more or less spindle to pine cone-shaped with many branches or small wartlike s~ les

F i g . 4. C a l c i t e s p i c u l e of the gorgonian L e p t o g o r g i a v i r g u l a t a . ( K i n g s l e y , u n p u b l i s h e d ) , x 8500 show partial extinction under the crossed nicols in a polarizing microscope> indicating they are polycrystalline aggregates (Kingsley and Watabe, unpublished).

Crystal Growth of Calcium Carbonate in the Invertebrates

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The c-axis seems almost parallel to the direction of elongation of the spicules. However, some of the immature spicules of Leptogorgia behave like single crystals with the c-axls parallel to the long morphological axis of the spicule. Spike-like spicules are found in the periostracum of certain bivalves (see Carter and Aller, 1975), in the cutlcles of polyplacophoran and aplachophoran shell plates (Beedham and Trueman, 1967, 1968, 1969; Schwabl, 1963; Haas, 1972), and they are all aragonite. They are either enveloped with the organic perlostracum or partially embedded within the outer prismatic layer. Echinoderms contain CaCO 3 spicules or ossicles in the dermis. They are of microscopic size and assume many different shapes from simple rod to wheels or perforated discs (see Hymann, 1955). Not much has been studies on the crystallography of these structures.

2.2.2. S p h e r u l i t i c aggregates and the r e l a t e d s t r u c t u r e . S p h e r u l i t e s are s p h e r i c a i aggregates of r a d i a t i n g c r y s t a l s . They are p r e s e n t in the s h e l l of cephalopod molluscs as a r a g o n l t e (Meenakshi and o t h e r s , 1974; Mutvei, 1964; Erben and o t h e r s , 1969). In the gastropod molluscs such as H e l i x p o ~ . ~ i a (Abolins-Krogis, 1968), Pomacea ~aludosa (Blackwelder and Watabe, 1977) or Ce~nemoralis (Watabe~llshed; Watabe and Vunkelberger, 1979), and the b1-~ve~Mytilus e d u l l s (Meenakshl and o t h e r s , 1973; Uozumi and Suzuki, 1978, 1979), a r a g o n l t e ~ p ~ t e s are formed at the e a r l y stage of s h e l l r e g e n e r a t i o n (Fig. 5). Aragonite s p h e r u l i t e s are a l s o r e p o r t e d at the e a r l y

Fig. 5.

Aragonite spherulltes formed at an early stage of Shell regeneration in the land snail Cepaea nemoralis. (Watabe). x 2700

s t a g e of skeleton formation in newly s e t t l e d l a r v a e of the r e e f - b u i l d i n g c o r a l P o c i l l o p o r a damicornis (Vandermullen end Watabe, 1973), and in the l a r v a l s h e l l s of the gastropod Hai i o t i s discus hannai (Iwata, 1980). C u t i c l e s of the decapods,

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e.g. Carcinus maenas (Digby, 1967, 1968) or amphipods (Reid, 1943) also contain

s p h e r u l i t e s , and the c a l c a r e o u s p o r t i o n (~ragonite) of c e r t a i n c o r a l l i n e sponges (HarCman, 1969) shows s p h e r u l l t e s t r u c t u r e . The process of spherulite formation is explained as follows: As seen in the n o r ~ l and regenerated shell of the cephalopod Nautilus macrom~halus (Meenakshi and others, 1974), doubly pointed needle crystals develop first; they then grow into stellate groups and finally into the spherulites. Alternatively, needle crystals form spindle shaped aggregates , at each end of which additional needles develop radially forming dumbbell structures; spherulite is completed by further development of needles in the middle region of the dumbbell (Blackwelder and Watabe, 1977). A morphological variation of the aggregate developed from the needle crystals is a short vertical cylinder, the walls ~f which are made of needles (Fig. 6). Such crystal aggregates are found on the inner surface of the

Fig. 6.

Cylindrical aggregates of aragonite needles formed in shell regeneration of Cepaea nemoralis. (Watabe). x 2400

gastropod L i t t o r i n a (Watabe, unpublished) and in the regenerated s h e l l s of the land s n a i l s Ce~aea n e m o r a l i s , and the sept,-, of Nautilus (Mutvei, 1972). Competitional growth of closely apposed spherulites leads to the formation of the spherulitic prismatic layer as seen in mollusc shells such as

archaeogastropod C i t t a r i u m pica (Wise and Hay, 1968a, b; Erben, 1971), cephalopod N a u t i l u s (Erben and o t h e r s , 1969; Mutvei, 1972; Meenakshi and o t h e r s , 1974), ligaments of the b i v a l v e s Mercenaria mercenaria and Brachydontes exustus (Mano and Watabe, 1979), tegument,-- s h e l l eyes of the plachophoran C h i t o n , 1972; Haas and Kristen, 1978), the skeletons of scleractinian corals (-~-~nes, 1970; Sorauf, 1970), and the cones of the sipunculid Lithacrosiphon surjanovae (watabe and Rice, unpublished). Advanced stage of regenerating shell of

Crystal Growth of Calcium Carbonate in the Invertebrates

107

Pomacea p a l u d o s a ( B l a c k w e l d e r and Watabe, 1977) shows a r a g o n i t i c s p h e r u l i t i c p r i s m a t i c l a y e r , and t h e s u p e r f i c i a l l a y e r o f t h e c h e i l o s t o m e b r y z o a n s such a s M e t r a r a b d o t o s s p . (Cheetham and o t h e r s , 1969), Stylopoma ~ a n d others ( S a n d b e r g , 1971) c o n t a i n s t r u c t u r e s p r e s u m a b l y d e v e l o p e d from a r a g o n l t e spherulites. C a l c i t i c s p h e r u l i t e s o r s t r u c t u r e s d e v e l o p e d from t h e s p h e r u l i t e s a r e r e p o r t e d i n t h e s k e l e t o n of t h e b r y o z o a Msmbranipora ( R i s t e d t , 1977, 1980), a x i a l r o d o f t h e a l c y o n a r i a n V e r e t i l l u m c~rnomorium (Ledger and F r a n c , 1978). In Veretillum the c-axls of calcite is parallel to the direction of elongation of each r a d i a l u n i t o f t h e s p h e r u l i t e s . J u d g i n g from t h e s c a n n i n g e l e c t r o n m i c r o g r a p h s and d i a g r a m s by Bourget (1977), s i m i l a r s t r u c t u r e s seem t o be p r e s e n t i n t h e c a l c i t e s h e l l p l a t e o f s e s s i l e b a r n a c l e s such a s B a l a n u _ ~ s a m p h i t r i t e and Chthamalus r h i z o p h o r a e . The c a l c i f i e d s h e l l l a y e r o f t h e a r t i c u l a t e b r a c h i o p o d s c o n s i s t s o f p r i m a r y and s e c o n d a r y l a y e r s o f c a l c i t e ( J o p e , 1977). W i l l i a m s ( s e e f o r e x a m p l e , 1 9 7 3 ) gave d e t a i l e d d e s c r i p t i o n o f t h e s e s t r u c t u r e s and i t a p p e a r s t h a t t h e p r i m a r y l a y e r r e p r e s e n t s one t y p e o f s p h e r u l i t i c p r i s m a t i c l a y e r . Spherulites are also r e p o t t e d i n t h e m i d d l e l a y e r o f t h e p e r i o s t r a c u m , on t h e r o o f a s w e l l a s on t h e f l o o r of a p e r i o s t r a c a l a n t r u m o f t h e f r e s h w a t e r m u s s e l , Amblema p l i q a t a ( P e t i t and o t h e r s , 1980); t h e c r y s t a l t y p e i s n o t known. 2.2.3. Vertical aggregates. An example of vertical aggregates is the prismatic layer of mollusc shells which is composed of more or less vertically (perpendicular to the shell surface) elongated columns of calcite or aragonite crystals (Fig. 7). Depending on the authors, the prismatic layer is divided into several structural varieties (see Kobayshi, 1971; Gr~goire, 1972; Carter, 1980a, b), and the simple prismatic defined by Carter (1980b) is an

F i g . 7.

Calcitic prismatic layer of the bivalve Pinctada fucata (~artensii). (Watabe, 1974). x 700

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N. Watabe

example of a typical vertical aggregate. The columns are enclosed by the sheath of organic matrix. Crystals within the columns are polycrystalline, and each seems to be also enclosed by the matrix membrane (Watabe and Wada, 1956; Wada, 1961). In many gastropods and marine bivalves, the prisms are made of calcite, and in others such as Unionacea and Trigonacea they are of aragonite (see Gr~goire, 1972). The myostracum, which is the region of mascle attachment in the shell, also shows a vertical structure and made of aragonite prisms. The orientation of the crystallographic axes vary, but commonly they are normal to the shell surface. However, they may be oblique to the surface in many cases (Gr~goire, 1972). In the freshwater gastropod Pomacea paludosa, regenerated shells form aragonite prisms after the formation of a follated layer and spherulitic prisms (Blackwelder and Watabe, 1977); and the egg shells show prismatic structure made of vaterite (Meenakshi, Blackwelder, and Watabe, 1974). In walls, particularly the frontal, of the cheilostome bryozoa, Schizoporella cornuta and Celleporaria vasans show columnar crystallites of calcite traversing the wall perpendicularly or at some high angles (Sandberg, 1971). Similar structure is also present in barnacle shells(Bourget, 1977) and articulate brachiopod shells (Williams, 1973). 2.2.4. Horizontally~ la.Tered aggregates: Typical of these aggregates are the nacreous layer and folxated layer (calcltostracum) of mollusc shells. Schmidt (1923) defined that the nacreous layer is the aragonitic innermost shell layer and composed of accumulation of numerous horizontal lamellae, each of which is an aggregate of tabular aragonite deposited on an interlamellar organic matrix (Figs. 8 and 9) (see also Gregoire, 1972; Watabe and Dunkelberger, 1979; Carter, 1980a for references). In most bivalves, the arrangement of crystal tablets in successive lamellae is either the "brick-wall" type (Fig. 8) or "stair-step" pattern. In gastropods, the cephalopod Nautilus, and the pelecypod Pirona, the crystal tablets are "stacked up" on each other, and each stack resembles the shade of a pyramid (Fig. ID). However, t h e s e pyramidal structures develop only at the surface layers and the major portions of the layer is similar to the bivalve nacreous layer. The interlamellar matrix sheets are also present in each layer, although the interlamellar spaces are not filled with crystals near the shell surface (Fig. 23) (Nakahara, 1979). Each aragonite tablet is polygonal to rounded (Figs. ii, 12), a few ~m to about IO Bm acros and is considered to be a single crystal or twins (Wada, 1961, 1972; Watabe, 1965; Erben, 1972, 1974). Each crystal may contain l a m e l l a r o r b l o c k d e n t r i t i c components, and an o r g a n i c m a t r i x ( i n t r a c r y s t a l l i n e matrix) is present between these components (Watabe, 1964; Mutvei, 1970). Optical and x-ray and electron diffraction studies show that the c-axis of aragonite is perpendicular to or nearly perpendicular to the inner growth surface of the shell (Schmidt, 1924; Wada, 1961; Watabe, 1965; Weiner and Traub, 198Oa, b). The b-axis is in the direction of horizontal growth of shells in bivalves (Schmidt, 1924; Omori, 1960; Wada, 1961; Watabe, 1965). I n g a s t r o p o d s t h e o r i e n t a t i o n was i m p l i c a t e d t o be s i m i l a r t o b i v a l v e s (Wada, 1961); however, Wise (1970) r e p o r t e d t h a t t h e y a r e a t random. I n t h e c e p h a l o p o d N a u t i l u s , t h e a - a x e s shows p a r a l l e l o r i e n t a t i o n (Wise, 1970).

The edge o f each l a m e l l a e e x p o s e d t o t h e i n n e r s u r f a c e o f t h e n a c r e o u s l a y e r i n d i c a t e s p a r a l l e l , s p i r a l , o r c o n c e n t r i c p a t t e r n s which a r e formed by t h e m a r g i n s o f e a c h a r a g o n i t e 1 - m g l l a (Watabe, 1955; Wada and H i r a n o , 1957; Wada, 1958). Wada (1969) r e p o r t e d t h a t t h e b - a x e s o f t h e a r a g o n i t e i n n e i g h b o r i n g s p i r a l s a r e p a r a l l e l w i t h one a n o t h e r and c o i n c i d e w i t h t h e direction of horizontal shell growth.

Crystal Growth of Calcium Carbonate in the Invertebrates

Fig. 8.

Fig. 9.

109

A "brickwall" type nacreous layer of Pinctada fucata, showing horizontal lamellae composed of aragonite tablets: x 3700

A decalcified vertical section of the nacreous layer of a pearl, showing interlamellar organic matrlxmembranes (horizontal lines) and intracrystalline m a t r i x ~ t e r i a l between the lamellae, cf. Fig 8. (Watabe, 1974). x 20000

II0

N. W a t a b e

Fig. IO.

A "stacked-up" type nacreous layer of the gastropod Turbo castanea. (Wise, Jr;, 1970). i .... x 2800;

2... x 5000;

3... x 180

Crystal Growth of Calcium Carbonate in the Invertebrates

111

Twinning of aragonite was reported in the nacreous layer of cultured pearls from Pinctada martensli (Watabe, 1955) and in the shell of monoplachophora Neopillna (Meenakshi and others, 1970). Recently, Mutvei (1977a, 1979, 1980) reported that each aragonite tablet of the nacreous layer of various mollusc shells showed complicated twinning patterns when etched with a glutaraldehyde acetic acid solutlon. In Nautilus and the gastropods Gibbula, Calli0stoma, Trochus, and Haliotis, the tablets are composed of a varying number of crystalline sectors which are polysynthetically twinned and separated by vertical organic membranes (Fig. 13). An organic accummulation is present in the central portion of each tablet. In the bivalves, Mytilus, Nucula, and Unio, Mutvei observed that each tablet is composed of two l a y e r ~ . 1 4 ) . The outer layer consists of four Polysynthetically twinned sectors, two of which

Fig. 13.

Aragonite tablets on the nacreous layer of the septal neck of Nautilus pompilus etched with sodium hypochlorite and glutaraldehyde-acetic acid, showing the central cavity and radial interspaces from which vertical organic membranes were removed. Crystalline sectors with striated surfaces are revealed by etching. (Mutvel, 1980). x 14000

112

N. Watabe

as Ostreidae, Pectinidae, and Anomiidae (see Kobayashi, 1971; Gr~goire, 1972; Carter, 1980a, b). This layer has an irregular, foliated structure made 6f thin and long calcite crystals which are in side-to-side contact making horizontal or semi-horizontal layers that overlap like shingles of a roof. (Fig. 15) (Tsujii and others, 1958, Watabe and others, 1958, Watabe and Wilbur, 1961). Similar to the nacreous layer, spiral patterns are observed

Fig. 15.

Calcite crystals on the surface of the foliated layer of the oyster Crassostrea virginica. (Watabe). x 18000

on t h e s e l a y e r s (Wada, 1968; K o b a y a s h i , 1971). The c - a x e s a r e normal to t h e h o r i z o n t a l p l a n e o f t h e c r y s t a l s , and t h e rhombohedral axes a r e o r i e n t e d i n a same d i r e c t i o n i n a r i m i t e d a r e a b u t d i f f e r from a r e a t o a r e a (Watabe, 1965; Wada, 1968). In Anomia l i s c h k e i , t h e rhombohedral axes a r e i n d i r e c t i o n s

radial from the center of the spirals (Wada 1968). The f51iated layer was also found at the early stage o~shell regeneration in Pomacea paludosa (Blackwelder and Watabe, 1977). Skeletons of the cheilostome bryozoans also contain calcitic lamellar structure roughly parallel to the frontal wall surface (see Sandberg, 1971; Ristedt, 1977, 1980). The secondary layer of cyclostome bryozoans reported by Soderquist (1968) and Taverner-Smith and Williams (1972) show a striking resemblance to the molluscan calcitostrac,--. Spiral growth of calcite has been found in Membranipora savarti (Ristedt, 1977) and cyclostomes (Taverner-Smith and Williams,

1972.-'~"--

C a l c a r e o u s t u b e s o f s e r p u l i d worms a r e composed o f s e v e r a l c o n c e n t r i c l a y e r s . Eupomatus d i a n t h u s (Muzi, p e r s o n a l comamnication) has t h e o u t e r a r a g o n i t i c ,

Crystal Growth of Calcium Carbonate in the Invertebrates

&

Fig. II.

P o l y g o n a l a r a g o n i t e t a b l e t s on t h e s u r f a c e o f t h e n a c r e o u s l a y e r of the marine gastropod L i t t o r i n a i r r o r a t a . ( W a t a b e ) . x 1600

F i g . 12.

Rounded a r a s o n i t e t a b l e t s on t h e s u r f a c e o f t h e n a c r e o u s l a y e r o f L. i r r o r a t a . (Watabe). x 2000

113

114

N. ~'~tabe

F i g . 14.

A r a g o n i t e t a b l e t s on t h e n a c r e o u s l a y e r of t h e b i v a l v e M ~ t i l u s e d u l i s a f t e r e t c h i n g . Note f o u r c r y s t a l l i n e s e c t o r s , two of which a r e l e s s s o l u b l e ( 1 . s . s . ) t h a n t h e o t h e r two ( h . s . s . ) . (Mutwei, 1980). x 8000

a r e much l e s s s o l u b l e than t h e o t h e r two i n t h e d e c a l c i f y i n g s o l u t i o n . The s u r f a c e of t h i s l a y e r shows c o n c e n t r i c growth l m e l l a e . The i n n e r l a y e r shows numerous p a r a l l e l l a t h s , i n d i c a t i n g a l a m e l l a r , p o l y s y u t h e t i c t w i n n i n g . These l a t h s a r e a s s o c i a t e d w i t h a n o r p h i c m a t r i x ; however, t h e t a b l e t s l a c k t h e c e n t r a l o r g a n i c a c c u m m u l a t i o n . Although t h e s e s t u d i e s gave a new i n s i g h t on t h e s t r u c t u r e of a r a g o n l t e t a b l e t s of t h e n a c r e o u s l a y e r , some of t h e r e s u l t s need to be interpreted with caution. For example, it is difficult to conceive that each t a b l e t e q u a l l y c o n s i s t s o f two s t r u c t u r a l l y d i f f e r e n t t y p e s of t w i n s - the upper c y c l i c and t h e lower p o l y s y n t h e t i c t w i n s . How do a l l t h e a r a g o n i t e t a b l e t s s w i t c h t w i n n i n g from one t y p e to t h e o t h e r u n i f o r m l y d u r i n g t h e i r growth? Etch f i g u r e s a r e d i s s o l u t i o n p a t t e r n o f c r y s t a l s and may r e f l e c t c r y s t a l symmetries (Buckley, 1955), b u t t h e y do n o t n e c e s s a r i l y r e v e a l a c t u a l s t r u c t u r a l u n i t s . I f the a r a g o n i t e t a b l e t s a r e composed of t w i n s , t h e a - or b - a x e s should n o t be o r i e n t e d i n p a r a l l e l d i r e c t i o n s . T h i s c o n t r a d i c t s w i t h t h e r e s u l t s of o p t i c a l and x - r a y and e l e c t r o n d i f f r a c t i o n s t u d i e s showing t h e p r e f e r r e d o r i e n t a t i o n of a r a g o n i t e as a l r e a d y m e n t i o n e d . However, t h e d i s c r e p a n c i e s c o u l d be due to s p e c i e s d i f f e r e n c e s . I n f a c t , Wada (1961) found t h a t t h e b - a x e s a r e randomly o r i e n t e d i n t h e cephalopod S e p i a . F u r t h e r s t u d i e s a r e needed t o a s c e r t a i n m o r p h o l o g i c a l and s t r u c t u r a l p a t t e r n s of a r a g o n l t e t a b l e t s i n t h e nacreous l a y e r . One o t h e r example of h o r i z o n t a l a g g r e g a t e s i s t h e c a l c i t o s t r a c u m ( f o l i a t e d l a y e r ) which i s p r e s e n t as t h e i n n e r m o s t c a l c l t i c s h e l l l a y e r of b i v a l v e s such

Crystal Growth of Calcium Carbonate in the Invertebrates

115

and t h e i n n e r c a l c i t i c l a y e r . The c a l c i t i c l a y e r i s d i v i d e d i n t o t h e o u t e r l a y e r of u n o r i e n t e d c r y s t a l s , and t h e i n n e r l a y e r of c r i s s c r o s s e d n e e d l e s . S p i r g r b i s b o r e a l l s has two l a y e r s , t h e o u t e r h e a v i l y c a l c i f i e d , and t h e i n n e r , f i b r o u s . and i n c o m p l e t e l y c a l c i f i e d l a y e r (Zur Loye, 1908). The tube of P r o t u l a c o n s i s t s of a l t e r n a t e l a y e r s of o r i e n t e d c r y s t a l s and the o r g a n i c m a t r i x ( S o u l l e r , 1891). D e t a i l e d s t r u c t u r e s of t h e s e l a y e r s a r e unknown, b u t some of them may show h o r i z o n t a l arrangement o f c r y s t a l s . F o r s m i n i f e r a u s a r e u n i c e l l u l a r organisms and the m a j o r i t y of them p o s s e s s calcareous tests. The i n n e r and o u t e r l a m e l l a e of the t e s t w a l l of t h e p l a n k t o n i c f o r a m i n i f e r a H a s t i E e r i n a and G l o b i g e r i n o i d e s a r e h o r i z o n t a l l a y e r s made of g r a n u l a r to p l a t y c a l c i t e , w h i l e t h e outmost c a l c i t e c r u s t shows p r i s m a t i c s t r u c t u r e composed of rhombohedral c a l c i t e c r y s t a l l i t e s (Hemleben, 1969a, b; B~ and Hemleben, 1970). 2.3.

Other T ~ e s of Aggregates

Granular structures are present in calcareous skeletons of many organisms. For instance, external surface of the lateral wall of cheilostome bryozoans such as Membranipora membranacea, and the internal portions of the primary layer of the cyclostome bryozoans such as Crisidia eburnea consist of minute calcite grains about 0.5 ~m across (Taverner-Smith a n ~ a m s , 1972). The homogeneous structure develops in the upper layer of the mollusc Limidae, and some layers of the Mytilidae consist of small aragonlte granules up to about 5 Um in diameter (see Gr~goire, 1972). I r r e g u l a r l y s t r u c t u r e d c a l c i t e was r e p o r t e d i n o s t r a c o d c a r a p a c e (Towe, 1972), and unorganized c a l c i t e n e e d l e s a r e p r e s e n t i n t h e i n n e r chamber w a l l of t h e planktonic formainfera, e.g. Peneroplis planatus (Hemleben, 1969a, b). The inner layer of the protoconch and pr~stomal shell of the archeogastropod Haliotls discus hannal contain acicular aragonite crystals inclined to the i n n e r s h e l - ~ ' - ~ f a c e - - ~ ' ~ w a t a , 1980). C r o a s - a c c i c u l a r s t r u c t u r e of a r a g o n i t e is a l s o r e p o r t e d i n t h e p r o t o c o n c h of P l e u r o t o m a r l a (Erben and Krampitz, 1972). Unorganized a g g r e g a t e s of a r a g o n i t e n e e d l e s a r e observed on the growth s u r f a c e of t h e p r i s m s of C i t t a r i u m p i c a (Wise and Hay, 19685) and i n t h e l a r v a l s h e l l s of t h e a r c h e o g a s t r o p o d Xances a n g u l a t u s and Buccinum undatum (Bandel, 1975), The c a l c i f i e d spear or d a r t (a r e p r o d u c t i v e s t r u c t u r e ) of many t e r r e s t r i a l pulmonate g a s t r o p o d s a r e up to about 1Omm long and t a p e r e d t o a p o i n t a t one end (Hunt, 1979; Tompa, 1980). In Helix pomatia, the dart is a four-bladed hollow spicule of calcium carbonate. The component crystals are randomly oriented minute aragonite, 0.5 ~m across and aggregated into sheets (Hunt,1979). The f i b r i l l a r s t r u c t u r e of m o l l u s c s h e l l s is composed of t h i n f i b r i l - l i k e a r a g o n i t e o r c a l c i t e c r y s t a l s w i t h i n d i v i d u a l l y c o n s t a n t d i a m e t e r from 1 to 2 ~m (MacClintock, 1967; C a r t e r , 1 9 8 0 a , b ) . Each f l b r i l p r o b a b l y e x t e n d s from the ventral to dorsal surface of the layer. All fibrils are inclined at angles ranging from 48 ° to 53° with the growth surfaces (Note: Kobayashi 1971, limited the fibrous structure to that made of aragonite crystals only). The inner shell layer of the euthecosomatous Pteropoda, a group of planktonic g a s t r o p o d s , e . g . C u v i e r i n a c o l u m n e l l a , c o n s i s t s of e l o n g a t e d and u n d u l a t i n g a r a g o n l t e f i b e r s about 0.2 ~m w~de a r r a n g e d i n a h e l i c a l s t r u c t u r e (B~ and o t h e r s , 1972). Curved c r y s t a l s of c a l c i t e a r e embedded i n c u t i c l e f i b e r s i n decapod c r u s t a c e a n integument ( T r a v l s , 1963; Mutvei, 1974, 1977b; Yano, 1980).

116

N. Watabe Aggregates of rhombohedral calcite are observed on the surface of the test of benthic foraminifera (Hemleben, 1969a, b; Be and Hamleben, 1970),egg shell of the terrestrial pulmonates (Tompa, 1976a, b), regenerated shells of Capaea nemoralis (see Fig. 20) (Watabe and Dunkelberger, 1979; Watabe, unpublished), and the statoliths of the cephalopod Sepia officinalis (Dilly, 1976) The most common structure in molluscan shells is the crossed lamellar structure (see Wise, 1971; Kobayashi, 1971; Gr~goire, 1972; Uozumi and others, 1972; Carter, 198Oa, b), and the majority are aragonitic. The smallest structural units are elongated rectangular rods (3rd order lamellae) about 2-6 um in thickness, which in turn are organized to form a rectangular block called first order lamellae. Rods of two adjacent second order lamallae are inclined in opposite direction with an angle of 45-50 degrees. The width of the first order lamellae varies with the species but somewhere around I0 to 20 ~m. According to Uozumi and others (1972) the rods are surrounded by organic matrix. Kobayashi (1971) reported that eosinophilic matrix is present around the rods, and basophilic fibers enclose the 2nd order lamellae. The crossed-lamellar aggregates are polycrystalline aggregates with a high degree of preferred orientation. Directions of crystallographic axes were determined in several species of polyplacophorans (Haas, 1972). It was found that the c-axes are oriented in the direction of bisectrix of the crystalline rods and b-axes are along the shorter horizontal axes of the lamellae (Fig.16).

Fig. 16.

A schematic diagram of the crossed-lamellar structure in the polyplacophorans, a,b,c: crystallographic axes of the aragonite, h, I, br: Vertical, long horizontal, and short horizontal axis of the first order lamella, resp. (Haas, 1972).

In reviewing the structures of the calcareous skeletons su----rized above, we will notice that in spite of the great diversities in the structures in different taxa, there are striking confor~ties in the crystalline patterns

Crystal Growth of Calcium Carbonate in the Invertebrates

117

building these skeletons. First, the sizes of crystals are very small and range from several hundred angstroms to several tens of micrometers across at the largest. Secondly, most of the structures are polycrystalline aggregates with preferred orientation, usually c-axes are oriented in a common direction; morphology of individual crystals are limited to a few variations. Thirdly, all the polycrystalline aggregates are associated with organic matrix. Fourth, single crystals occur as calcite (high magnesium) and only in the sponges and echinoderms without or little association with organic matrix. Apparently, the processes of formation and growth of crystals are very well regulated in the invertebrate systems, and the conditions for crystallization may be uniform among many organisms even though they belong to different taxa and their overall skeletal morphologies are different. In order to understand the processes of crystallization, we shall examine the sites of formation and growth of CaCO 3 crystals in various organisms in the next sections. 3.

SITES OF FORMATION AND GROWTH OF CaCO 3 CRYSTALS

In biological systems, the CaCO 3 crystals develop and grow intracellularly or extracellularly (Watabe, 1974, 1981; Simkiss, 1976; Wilbur, 1976, 1980; Wilbur and Simkiss, 1979). In addition, recent studies (Kingsley and Watabe, unpublished; Sbimizu and Yamada, 1980) have shown that in some organisms an initial intracellular crystal formation is succeeded by an extracellular deposition at later stage of growth. 3.1.

Intracellular Sites

3.1.1 Within single cells. No single crystal structure has been found to be associated with this type of formation. Examples of this type are observed in most of the spherule formations in various organisms (see section 2) in which amorphous CaC03, vaterite, or calcite crystals are formed in vesicles or vacuoles frequently associated with the Golgi complex, e.g. in Pomacea paludosa (Kurtz, 1975; Watabe and others, 1976) and in diplopode insects (Hubert, 1979); or with the endoplasmic reticulum, e.g. in the protozoa, Poronodon (Andr~ and Faur~-Fremiet, 1967) and in the homopteran insect Philaenus squmaris (Gouranton, 1968). As seen in the spherules of Pomacea, the organlc matrix is formed within the vesicles or vacuoles prior to the cryb=al formation. The spicules of acochlidiacean molluscs, e.g. H edylopsis sp. are formed in the spicule forming cells localized in the connective tissue underlying the epidermis (Rieger and Sterr, 1975b). Calcite form,~:ion is observed within the mitochondria of the calciferous gland of the earthworm, Lumbricus terrestris (Crang and others, 1968). These crystals are extruded into the gland lumen, where they develop into the calcareous concretions. On the contrary, Nakahara and Bevelander (1969) reported that the spherules of the calciferous glands are not present in the cell proper, but they appear in diverse state of mineralization in the extracellular gland cevity; but the origin of the spherules are reported to be the Golgl granules. Aggregates of calcite crystals (150-200 ~m on a side) are formed within a matrix material in the multivesicular bodies of calcium secreting gland of the serpulid worm, Pomatocerus caeruleus during the tube regeneration (Neff, 1971). They are also secreted into the lumen, and possibly are utilized for building tubes. It should be mentioned that this type of crystal formation is not limited to the invertebrates. In the unicellular calcareous algae, Elimian~a huxleyl (Wilbur and Watabe, 1963) and others (see Blackwelder and others, 1979)', coccolith (calcareous discs made of calcite crystalites) are formed within the Golgi vacuoles.

118

N. Watabe

3.1.2. In s~rncytia. Crystal formation in syncytia has been reported only in the echinoids. Larval spicules (see Okazaki and others, 1980) and teeth (Kniprath, 1974) formation takes place within a vacuole in the cytoplasm formed by fusion of several cells. Repair of test plates is also reported to take place in a similar fashion (Vocisano, 1971) (but see Shimizu and Yamada, 1980). It may be worth mentioning that all of the crystals proven to be formed intracellularly are either amorphous CaCOg, vaterite, or calcite and no aragonite formation has yet been confirmed. The site of formation of aragonite spherules in Nerita is unknown. The only exception may be the aragonite spicule of the compound ascidians reported by Lafargue and Kniprath (1979) and Kniprath and Lafargue (1980) in which the crystals are formed within the "scleroblasts" vacuoles of the lateral organs of the thorax. However, their mlcrograph suggests the spicule-bearing vacuoles are outside of the "Scleroblasts" and further examination on the sites of crystal formation in this organism seems warranted. 3.2.

Extracellular Sites

3.2.1. Within the animal body. This type of formation is found in the calcareous sponge, Sycon ciliatum (Ledger and Jones, 1977), in which a spicule is formed in an intercellular cavity enclosed by a group of sclerocytes. The cavity contains an organic sheath which envelopes the spicule. Spine regeneration in the sea urchin, Strongylocentrotus purpuratus is reported to take place extracellularly in the dermis (Heatfield and Travis, 1972, 1975). A thin cytoplasmic extension of calicoblasts envelope the broken microspines and supplies materials for the spine growth (but, see Shimizu and Yamada, 1980). The site of formation of holothuroidean that a small rod or disc appears by the more mesenchyme cells (see Hymann, 1955) formation. However, no ultrastructural details are not known.

spicules is not very clear. It is reported secretion of the aggregates of two or which implies extracellular type of studies have been reported to date and

The spherules in the mantle of the bivalve mollusc Mercenaria mercenaria are formed in the intercellular space (Istin and Masoni, 1973). In the posterior caecum of the amphipod crustacean Orchestia caviniana, the basal and lateral plasma membranes of the epithlia cells invaglnate to form many chambers in which the spherules develop (Graf, 1971). Calcite rods of the alcyonarian coral Veretillum c~nomorium are formed in the cortex by the secretion from the epide~'~ (Ledger and Franc, 1978). The spicules of caudofoveates are secreted by the epidermis and embedded or connected in the cuticla (Rieger and Sterrer, 1975b); therefore, they do not seam to be of the intracellular origin as Schmidt (1924) reported. The turbellarians Florianella, Bertiliella, Acanthiella, and Carcharodorh~nchus contain spicules in the basal lamina underlying the epidermis {Rieger and Sterrer, 1975a) and appear to be extracellular products. 3.2.2. Outside of the animal body. Most of the calcium carbonate structures such as scleractinian coral and bryozoan skeletons, brachiopod and mollusc shells, serpulid worm tubes and sipunculid cones, and arthropod skeletons are formed outside of the animal body through the secretory activities of epidermal tissues. S e v e r a l c o n t r o v e r s i a l o p i n i o n s have been p u t f o r t h c o n c e r n i n g t h e i n i t i a l s i t e s of scleractinian coral calcification. However, i t i s now g e n e r a l l y b e l i e v e d t h a t the c a l c i f i c a t i o n takes place at the o u t e r surface of the c a l l c o b l a s t i c

Crystal Growth of Calcium Carbonate in the Invertebrates

119

epithelium and it is an entirely extracellular event (see ¥amazato, 1974; Johnston, 1980). Although Hayes and Goreau (1977a, h) and Goreau and Hayes (1977) claimed to have obtained evidence of intracellular aragonite crystal formation in Scleractinia, no solid proof of presence of aragonlte (or calcium carbonate c~ystals) have been presented. The calcareous tests of foraminifera are also formed extracellulary through the pseudopodial extension of the cell. Angell (1965) followed the process of calcification in the benthic foraminifera Rosalina floridana which was smmnarized by Pautard (1970) as follows:

v

• i~ ii'i

~~ ~ :~

~ / /

F i g . 17.

~-

U l t r a s t r u c t u r a l p r o c e s s of c a l c i f i c a t i o n Rosalina floridana. ( P a u t a r d , 1970).

i n the f o r a m i n i f e r a

l,

A thin cyst is first formed by the cytoplasm (Fig. 17a)

2.

The new chamber a n a l a g e i s made by p s e u d o p o d l a upon which t h e chamber w a l l i s b u i l t ( F i g . 17b).

3.

The l i n l n g of chamber i s c o n s t r u c t e d ( F i g . 1 7 c ) .

4.

C a l c i f i c a t i o n a p p e a r s t o commence by t h e a p p e a r a n c e of numerous v e s i c l e s and vacuoes ( F i g . 17d).

5.

The vesicles flow through the aperture of the new chamber, forming a vacuolated sheath which settles down to form the calcite area in the wall (Fig. 17e).

6.

The cytoplasm retreats in the lower parts of the chamber, leaving the final wall complete (Fig. 17f).

120

N. Watabe

The perinotum spines of the placophora Lepidochitona cinera were believed to be formed "quasl-intracellularly" in the epithelium (Has~, I-9~6). However, later studies in Acanthopleura granulata indicated that the spines developed in the cavity formed'by invaginations of the epithelium, and they are entirely of extracellular origin (Haas and Krlesten, 1977). An interesting observation was reported by Haas and his colleagues (1980) on the early shell plate formation of the larvae of Lepidochitona. The cells of the groove complex of the dorsal epithelium become broader and higher at certain larval stage, and the cells at the outer peripheries of the groove extend a few of their microvilli laterally. These cytoplasmic extensions from two cells meet and form a chamber, in which primordial shell develops. Although the site of crystal formation is mostly isolated from the environment, it is still extracellular. 3.2.3. Intracellular and extracellular formation. Recent studies by Shimizu and Yameda (1976, 1980) have shown that both the spine and test regeneration of the sea urchin Stron~ylocentrotus intermedius take place first intracellularly and then later extracellularly. As shown in Fig. 18, initial crystallites of several se¢ondary ,¢Jerocyte

2::::: °..'

Fig. 18.

P2::;

Schematic illustration of the general course of crystal formation in regenerating test and spine in sea urchins. C: crystal. (Shimizu and Yamada, 1980).

micrometers long develop in smell vacuoles of the primary sclerocytes in close association with the Golgi complexes and mitochondria. As the crystals grow larger, the vacuoles also enlarge and the vacuole membranes fuse with the cell membranes. Finally, the membranes rupture and the crystals become extracellular. Further growth of the crystals is aided by another type of cells (secondary sclerocytes) which come to cover the crystal surfaces with their elongated cytoplasmic extensions. The next step is the fusion of these crystals into stereomal islands, which seemed to merge into a larger stereom. Finally, the stereom develops into a fully calcified regenerated skeleton continuous with and similar to the normal one. The results imply that the regenerates become incorporated into the old spine (or test) as the integral parts of the single crystal. In order for this type of crystal accretion to be accomplished, the different individual crystals

Crystal Growth of Calcium Carbonate in the Invertebrates

121

must be aligned with all the crystallographic axes parallel with one another. Since the major portion of the "regenerate" crystals are enclosed by the primary and secondary sclerocytes, the alignment should also involve morphological alteration and/or relocation of these cells. Also, some dissolution must take place at the surfaces of crystals to be fused together. At present, ~here is no information available concerning these points. Spicules of Leptogor~ia virgulata was found to be initially formed in the Golgi vacuole of the scleroblasts situated in the mesoglea. Similar to the process described in the regenerating skeletons of the sea urchin, the vacuole expands with the advancement of the spicule growth and open with a narrow channel to the extracellular space of mesoglea (Fig. 19) (Kingsley and Watabe, unpublished). Each crystal continues to grow in the extracellular cavity

Fig. 19.

A t r a n s m i s s i o n e l e c t r o n micrograph showing the s p i c u l e growth in the s c l e r o b l a s t of Leptogor6ia v i r g u l a t a . The s p i c u l e (S) i s in c o n t a c t with the e x t r a c e l l u l a r space (arrow). (Kingsley, unpublished), x 7500

surrounded by the s c l e r o b l a s t s cytoplasm. Although the a l c y o n a r i a n ~ e n i l l a r e n i f o r m i s was reported to have i n t r a c e l l u l a r s p i c u l e s (Dunkelberger and Watabe, 1974), our l a t e s t i n v e s t i g a t i o n suggests the formation and growth of spicules in this organlsmmay be similar to those of Leptogorgia (Watabe, unpublished).

N. Watabe

122

4.

NUCLEATION AND GROWTH OF CaCO 3 AND ROLES OF ORGANIC MATRIX

Formation of new crystals from solution, in which no such crystals existed previously, involves two phases: crystal nucleation and crystal growth. Nucleation is considered to be achieved by a stepwise addition of single molecules, atoms, or ions, and eventually a lattice is formed. Many of these sub-nuclel are unstable and redlssolve. Those survived grow beyond a certain critical size and are stable, and the nuclei for further crystal growth are formed (see Glimcher, 1959; Mullin, 1961; van Hook, 1963). Nucleation may take place in the presence of foreign materials (heterogenous nucleation), or spontaneously (homogenous nucleation). In biological systems, the solution from which CaCO 3 crystallizes out, e.g. the extrapallial fluid between the mantle and shell of molluscs, contains and/or is in contact with many inorganic and organic materials (see Table I) and the nucleation can be considered to be of heterogenous. It is generally accepted, at least conceptually, that the organic matrix serves as the sites of nucleation of CaCO 3 crystals or as the substrate on which the crystal nucleii are deposited. Evidence for the formation of CaCO 3 crystals (calcite) on the organic matrix was shown in the bivalve Craesostrea virginica (Wilbur and Watabe, 1967) and the initiation of crystals in 6he organic matrix has been observed in many organisms. The examples include the spicules of alcyonarian corals, 4DunkelberEer and Watabe, 1974) 4Kingsley and Watabe, unpublished), serpulid polychaete worms (Neff, 1971), arthropods (Travis, 1960, 1963; Yano, 1970, 1972, 1980), and molluscs (see Watabe, 1974; Wilbur, 1976; Watabe and Dunkelberger, 1979; Wada, 1980). The mechanism of the induction of crystal nucleation by the organic matrix is not very well understood. In molluscs, the organic matrix consists of variable proportions of soluble and insoluble fractions, both composed of proteins and carbohydrates with varying compositions by the taxa, habitat, and the accompanying mineral polymorphs (see Wilbur and Simkiss, 1968; Gr~goire, 1972; Krampitz and others, 1976; Weiner, 1979; Samata and others, 1980 for references). The insoluble fraction constitutes a major portion of the matrix and is present between the crystalline layers and also surrounds the crystals. The soluble matrix seems to be sequestered within the crystals (Wilbur and Simkiss, 1968), or may be present in the perforations in the interlamellar (insoluble) matrix of the nacreous layer (lwata, 1975), or at the outer and inner surfaces of the insoluble matrix (Weiner and Traub, 198Oa). The soluble matrix was found to contain Ca-binding components in several bivalves and gastropods 4Crenshaw, 1972b; Krampitz and others, 1976, 1977; Samara and others, 1980). However, the exact sites of the binding are yet to be identified. One of the possible sites frequently discussed is the carboxylic groups of aspartic and glutamic acid residues (see Wilbur, 1976); however, Crenshaw 41972b) and Krampitz and others 41976) maintain that most of these residues are present as amides and would bind little or no calcium. Samara and others 41980) found that the aspartic and glutamic acid residues in the Ca-binding fraction in Crassostrea gigas were relatively low to be considered to be the candidates for the binding. On the other hand, Crenshaw and Ristedt (1975, 1976) identified Ca-bindlng sulfated polysaccharide sites in the (insoluble?) matrix covering the aragonite crystals in the nacreous shell layer of Nautilus pompilus. (The material may be a sulfated Ca-binding glycoproteln similar to the one isolated from Mercenaria mercenaria by Crenshaw 41972b). They have proposed a hypothesis that the Ca-binding sites bring about a local increase in calcium and bicarbonate through the process of ionotropy, resulting in the precipitation of CaCO 3 crystals.

Crystal Growth of Calcium Carbonate in the Invertebrates

,,,~ r~ •

•

O =

0 ~J 0

=

0 ~J

F~

o,~

"0

v-q m 0

+I ==

u~

123

124

N. Watabe

The molluscan organic matrix is also proposed to serve as a template for epitaxial formation of crystal nucleii. It was shown experimentally that the matrix (mostly insoluble) from calcitic or aragonitic shells induced, although with a low success rate, calcite or aragonite crystal formation, respectively (Watabe and Wilbur, 1960; Wilbur and Watabe, 1963). In more recent studies (Watabe in Watabe and Dunkelberger, 1969) the organic matrix was obtained by decalcification of aragonitic or calcitic shells in chromium sulfate or EDTA solution, containing cetylpyridinium chloride which presumably retain soluble matrix and/or acid mucopolysaccharides. The matrix was used as the substrate for shell regeneration in the land snail Cepaea nemoralis which deposits a mixture of calcite and aragonite in normal regeneration (Fig. 20). It was found

Fig. 20.

Mixed growth of rhombohedral calcite and spherulitic aragonite in shell regeneration of Cepaea nemoralis. (Watabe). x 3000 that in one case out of ten only calcite was found on the matrix from calcitic

Crystal Growth of Calcium Carbonate in the Invertebrates

125

shells, and similarly only aragonite was found on the aragonlte matrix (Fig. 21).

Fig. 21.

Aragonite t a b l e t grown on the organic matrix obtained from a r a g o n i t l c s h e l l ( H a l i o t i s nacreous l a y e r ) in s h e l l r e g e n e r a t i o n of Cepaea n e m o r a l l s . (Watabe). x 6300

Moreover, the morphology of those crystals was different from that of the normal shell regenerates and was rather similar to that of the shells from which the matrix was obtained. Of course, those results are far from conclusive, hut at least they suggest the epitaxial role of organic matrix in inducing crystal polymorphism in mollusc shells.

The template concept is a l s o suggested by Welner and Traub (1980a, h ) . They found a well-defined spatial relationship between the insoluble component of the matrix and crystallographic axes of aragonite in Nautilus repertus. It was assumed that the intervening soluble matrlx is also aligned, and If t~ematrix surface has the repeating distance between the negatively charged aspartic acid residues which corresponds to the Ca 2+ - Ca 2+ distance of the b-axis of aragonite, or some multiple of it, the surface would provide a potential nucleating zone (Fig. 22a). On the other hand, when the distances between the •

•

,

~.

126

N. Watabe

D ~--8 ~,----~ ~

6xg.0A= 48.0A Ca-Co distance along b axis

~Ca

C~a

Ca

Ca

~

C~a

C~a

Aragonil.e sur.f-ace

e

~

~

~

"

I,

~

,

,

Organic mat'r ix surface

7 x6.9A -48.3 A

Fig. 22a.

A schematic diagram of hypothetical crystal nucleating surface - the distance between negatively charged aspartic acid residues along the polypeptide chains corresponds to

, = i t i p l e (the diagr=.shows six =

ltiple) of the

- Caz+ d i s t a n c e o f 8 X a l o n g the a r a g o n i t e ~ a x i s . (Weiner am] T r a u b , 1980b).

negative charge do not correspond to the b-axis distance (Fig. 22b), the matrix surface may inhibit the crystal growth.

~eeA-~

@

®

®

®

@8oo0d

No correspondence Y / / / / / / / / / I / / / / / / ~ Aragonite surface I-- 8.0~,--I Fig. 22b.

Ca-Ca Distance atonq b axis

A hypothetical crystal growth inhibiting surface. The distances between negatively charged aspartic acid residues do not correspond to Ca 2+ - CaZ÷ distances along the aragonite b axis. (Weiner and Traub, 1980b).

Another function assigned to the organic matrix is the formation of "compartment" in which the crystals develop. This was proposed by Bevelander and Nakahara (1969) in the nacreous layer of mollusc shells, and later applied also to the various calcareous structures (Nakahara, 1979; Bevelander and Nakahara, 1980). The requiremant for the "compartment theory" is the presence of preformed and well-deflned envelopes of organic matrix. Therefore, the mass of organic matrix in which the crystal nucleation and gro~ch take place as in Renilla and other organisms is not the compartmont. An example of the f o r m a ~ of the compartment and the crystals g r a p h is described for the development of the prismatic layer of a mollusc (Bevelander and Nakahara, 1971), i.e. an electron-dense lamella is elaborated in the proximal region of the outer surface of the outer mantle fold, and fragments of the lamella become detached and migrate to a chamber bounded by the periostracum; there, these lamellar fragments form envelopes, within which crystal initiation and growth occur. The envelopes have been shown to be present in the early crystal formation of: spherules of the calciferous gland of the earthworm; aragonite needles of the alga Halimeda; and the otoliths of two-day-old mouse (Bevelander and Nakahara, 1980). However, Erben (1972, 1974) and Erben and Watabe (1974) found no evidence of preformed

Crystal Growth of Calcium Carbonate in the Invertebrates

127

compartments in the nacreous layer of bivalves. Although organic matrix materials were observed in the environment in which the crystal growth takes palce, they were by no means the well-defined envelopes. When the crystals are brought in contact with one another by growth, the matrix materials become sandwiched between the crystals. When the crystals are decalcified at this stage, the matrix shows the configuration of envelopes surrounding the crystals. In fact, the glutaraldehyde fixative utilized in those studies have been known to dissolve CaCO 3 crystals (e.g. Ledger and Jones, 1977). Dunkelberger and Watabe (unpublished) also found that many of the calcite crystals at the early stage of development of juvenile fish otoliths were decalcified after the glutaraldehyde fixation leaving the envelope-t~pe matrix structure, giving a false impression that the envelopes were formed prior to the crystal development. The mechanism of formation of the "stack-up"-type nacreous layer in gastropods (see Fig. iO) have been explained by Wise (1970), Uozuml and Togo (1975), and Nakahara (1979) by the compartment theory. According to Nakahara's electron microscope study (1979), many parallel sheets of interlamellar matrix are present without the crystals in the nacreous layer (Fig. 23). It is proposed that the

Fig. 23.

A vertical section of the "stacked-up" nacreous layer of Crystal stacks (C) and lamellar sheets of organic matrix (S) are shown. (Nakahara, 1979)i. x 8400

Tegula pfeifferi.

successive compartments are formed in the spaces between these sheets prior to the crystal formation. However, the structure can develop in the following sequence without the preformation of the compartment (Fig. 24a) (Wat~be, unpublished)

128

Watabe

N.

(a)

(b)

~" "':'~ ; ~ ' : " : ' " : ; : ' " t : ' : ' " ' : ' " ' : ' " " : ' ,

M-i

."~:"..".';."¢':'~ . ' , ' . . ' . . : ' . ' r :

. T ' : :~... M-I

2

• ' " : "" : ' " " : - "

"'"-'~" ; ' ~ ' "

" "" " ' " ": ~" "': " " " M"

•; •"~.'-_iiJ ' ~ ~ - . ~' ! .'-.;.'".' '-"- "."" "

:f

C-I

Y : ' ~ ' f C-I

J;%(~C-I

'

Fig. 24.

'

..

I;:M. I

M-2 M-I

M-2

A schematic diagram showing the process of nacreous layer formation. (Watabe). a.

"Stacked-up" nacreous layer.

b.

"Brick-wall" nacreous layer.

I.

An interlamellaf matrix sheet (M-l) is deposited with non-lamellar matrix over it.

2.

Ca 2+ and HCO~- ions are secreted onto the matrix region, and CaCO 3 crystals (C-I) are fo~med on the matrix (M-I),

3.

Before the crystals (C-l) grow larger and come in contact with one another, another matrix (M-2) is laid down

4.

The crystals (C-2) are formed and grow over the matrix (M-2). (M-3) is laid dow

5.

The crystals (C-3) develop over (M-3).

As shown by Erben (1972), Wada (1972), Mutvei (1977, 1979, 1980), and Nakahara (1979), the crystals develop over the existing ones (C-I) or (C-2) possibly by overgrowth through the perforations in the interlamellar matrix. Also, new crystals may be nucleated and grow on the interlamellar matrix• At the same time, the ions diffuse through the matrix (M-2) or (M-3) (Weiner, personal communication) and the crystals (C-l) or (C-2) continue to grow horizontally. The overgrowth on the horizontal surface of aragonite is evidenced by the continuation of the crystals from one layer to the upper layer shown by Wada (1972) and also by the frequent occurrence of corner and edge growth of nacreous

Crystal Growth of Calcium Carbonate in the Invertebrates

129

aragonite (wada, 1961; Watabe, unpublished). Depending on the extent of horizontal growth, the c r y s t a l s of neighboring s t a c k s would show the offset interlocking (Fig. 10.2). The vertical growth of the crystals will become inhibited by the overlylngmatrix layer (Watabe, 1965; Wilbur, 1972; Crenshaw and Ristedt, 1975, 1976; Weiner and Traub, 198Ob), and a relative uniformity in the thickness of the crystals is achieved. Judging from the Nakahara's micrograph (Fig. 23) it will be after the deposition of about IO interlsmellar matrix before the crystals come to fuse together to form a complete crystall~ne layer.

In the formation of the " b r i c k - w a l l " type nacreous l a y e r , the deposit;ion of the i n t e r l a m e l l a r matrix ( e . g . M-l) and the subsequent formation of c r y s t a l s (C-l) are similar to the "stack-up" type. However, the next episodic deposition of the matrix layer (M-2) may occur after or about the same time when the crystals (C-I) come in contact with one another (Fig. 245). The next crystals (C-2} also grow over the (C-l) by overgrowth or by nucleation on the matrix and form a crystalline layer. Thus, the main difzerence in the mechanism of the formation of the two types of the nacreous layer would be the frequency of the matrix layer deposition relative to the rate of the crystal growth. As described previously, the echinold skeletons and the calcareous sponge spicules do not appear to contain an appreciable amount of organic matrix within the crystals. However, at least at the initial stage of the ehhinold crystal formation, an organic material is observed to be incorporated into the vacuoles or extracellular cavities and is implicated to play a role in calcite nucleation (Gibbins and others, 1969; Kniprath, 1974; Heatfield and Travis, 1975; Shimizu and ¥amada, 1980). In the sponge, ~ c i l i a t u m , Ledger end Jones (1977) observed that a primordial crystal was formed wlthin a space enclosed by an organic sheath in the intercellular cavity. Those authors maintain that the sheath neither serves as an epitactic membrance nor "compartment" or "mold". Inorganic ions diffuse through the sheath and deposit on the crystal. Thus, the crystal grows and stretches the sheath by the growth. The crystal type and orientation do not seem to be determined by the m-trix, but rather by the composition of the liquid from which the crystal forms and by the shape of the space of the crystal growth. However, although the sheath seems to be nonoriented and stereochemically unspecific, it could induce nucleation within itself (Ledger and Jones, 1977). The spherulites of the axial skeleton of Veretillum cynomorium are initiated by the formation of small(dalcite) crystals in the inferior tip of the rod

(Ledger and Franc, 1978). Sometimes, these c r y s t a l s are a s s o c i a t e d with c e l l u l a r d e b r i s . The c r y s t a l s develop into nodules, from which c o l , - / - a r c a l c i t e grow r a d i a l l y to form s p h e r u l l t e s . I n t e r e s t i n g l y , c o l l a g e n f i b e r s are associated with the calcite crystals in this organism. The fibers are embedded in the calcite but not impregenated with it, and are not responsible for the initial nucleation of mineral; the crystallographic orientation of the calcite does not seem to be related to the fiber orientation. The association of collagen with calcium carbonate crystals is very unique and found only in axiferous octocorals (Goldberg, 1976; Watabe and Dunkelberger, 1979). Nevertheless, even though the skeletal rod is a calcified collagenous tissue, it is not a calcified collagen as such (Ledger and Franc, 1978). Thus the actual sites of nucleation of the crystals are not clear, and further studies are warranted.

In Renilla r e n i f o r m i s (Dunkelberger and Watabe, 1974) or Leptogor~ia v i r g u l a t a (Kingsley and Watabe, unpublished) the c a l c i t e c r y s t a l s are formed in the fibrous matrix within a vacuole. Subsequent growth of spicules are accomplished by deposition of alternating concentric layers of crystals and matrix. In Renilla the diameter of individual crystals at an earlier stage of spicule growth

130

N. Watabe

are about 0.4 Bm but that at the later stage was about 0.2 ~m. The difference in the crystal size accompanies the deposition of different organic matrix in the s p i c u l e v a c u o l e (Watabe end D u n k e l b e r s e r , 1974). 5.

FACTORS CONTROLLING THE CRYSTAL CHARACTERISTICS

We have a l r e a d y d i s c u s s e d t h e p o s s i b l e

roles of the organic matrix in i n i t i a t i o n o f c r y s t a l f o r m a t i o n , i n h i b i t i o n o f c r y s t a l growth, and i n c r y s t a l p o l y m 0 r p h i s m . The m a t r i x may a l s o a f f e c t t h e c r y s t a l morphology; e . g . e i g h t d i f f e r e n t forms o f a r a g o n i t e c r y s t a l s were found i n t h e s e p t a l - n e c k n a c r e o u s l a y e r o f N a u t i l u s , and e a c h c r y s t a l was a s s o c i a t e d w i t h d i f f e r e n t amounts of organic matrix. The c r y s t a l s i n a r e g i o n low i n m a t r i x were e i t h e r d e n d r i t i c o r t e n d e d t o be p o r o u s , end t h o s e i n m a t r i x - r i c h r e g i o n s o f t e n had a g r a n u l a r s t r u c t u r e (Mutvei, 1972). G r ~ g o i r e (1973) a l s o r e p o r t e d a d i f f e r e n c e i n t h e o r g a n i c m a t r i x s t r u c t u r e i n d i f f e r e n t r e g i o n s o f t h e s e p a t a l n e c k , which substantiates the crystal-matrix relationship just described. Another factor influencing the crystal morphology is reported to be the microtopography of the surface of the structure on which crystals develop, and a mold and cast relationship seems to exist between the crystal'form and the substrata structure (Taylor and Kennedy, 1969; Meenakshi and Donnay and others 1974). Environmental factors such as temperature, salinity, ionic concentrations etc. have been shown to affect the crystal formation (see Wilbur, 1964). In molluscs, temperature and salinity modify the shell mineralogy and there is an inverse relationship between the calcite content and the average water temperature. This is also found in cirrlpeds, octo¢orals, serpulids, and bryozoans (see Lowenstam, 1963, 1964). Isolated patches of calcite within aragonite shells of Protothaca staminea increased in the water below lO°C (Carter and Eichenberger, 1977). Higher salinity also seems to cause a higher calcite to aragonite ratio in molluscs (see Carter, 198Oa). However, since aragonite and calcite are usually present in each discrete shell layer in molluscs, the increase or decrease of each mineral is a reflection of changes in the formation of a particular layer (see Kennedy and others, 1969). Thus, the environmental effects od skeletal mineralogy appear to be indirect and mediated through the metabolisms of the organisms rather than the direct physico-chemical control by the environment. Shell regeneration in Pomacea paludosa is initiated by the formation of calcitic layer, followed by the deposition of aragonite layers (Meenakshi and others, 1975; Blackwelder and Watabe, 1977). The ratio of aragonite to calcite was found to increase at higher temperature; however, this is because the organisms resume normal aragonlte shell layer formation faster than at lower temperatures (Watabe, 1981). The higher percentage of aragonite observed by Wilbur and Watabe (1963) at higher temperatures in the regenerated shells of the freshwater snails Viviparus intertexus could also have been the results of higher percentages of normal shell deposition at those temperatures. A variety of trace elements have been found to be present in the skeletal structures. Onuma and others (1979) found that the relationship between the partition coefficient of elements in the inner shell aragonite of the marine bivalve Pinctada fucata and the ionic radii follows a hyperbolic curve in the case of divalent Io~-~-,but not for monovalent ions (Fig. 25).

Crystal Growth of Calcium Carbonate in the Invertebrates

131

Ca ld

5r .... lo

E U

o uE' 1 0

7

.9

Cu o

Na

Znl

•

K

Mg

®

'1~ 06

|

i

i

i

I

I

|

i

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

Radius

(A)

Ionic

F i g . 25.

Partition coefficient-ionic radius diagram for aragonite i n n e r s h e l l l a y e r and e x t r a p a l l l a l fluid of the bivalve Pinctada fucata. (Onuma and o t h e r s , 1979)

The partition coefficient defined was the ratio of concentration of an element in the shell to that in the extrapallial fluid. The curve implies that those cations (Sr 2+, Mn Z+, Fe 2+, Zn 2÷, and Mg 2+) occupy regularly the lattice sites in the crystal structure of aragoniteo (Mg is known to be present in MgCO 3 which make~z÷a.solid solution wlth" calclte. (See Kitano and others, 1976)) The value for Cu is higher than the curve, which may indicate that the excess amount of the element exists in the organic matrix of the shell. On the other hand, monovalent ions Na + and K + are considered to be inclusions such as carbonates and/or amorphous compounds (Onuma and others, 1979). However, these results are for relatively high concentrations of minor elements, and it is not known whether the elements at low concentrations are bound to the organic matrix component, or incorporated into the CaCO 3 crystal lattice, or absorbed on the crystal surfaces (Rose~herg, 1980).

Magnesium r o n t e n t i s r e p o r t e d t o be d e p e n d e n t upon t h e m i n e r a l o g y and t a x o n o m i c r e l a t i o n s h i p o f t h e o r g a n i s m s ( C l a r k and W h e e l e r , 1917; Chave, 1954), and i s h i g h e r i n c a l c l t l c t h a n i n a r a g o n l t l c s k e l e t o n s . Water t e m p e r a t u r e and s a l i n i t y a r e a l s o two o f t h e f a c t o r s and t h e Mg c o n t e n t i n c a l c i t e i s h i g h e r a t h i g h e r t e m p e r a t u r e s (Lerman, 1965; K i t a n o and o t h e r s , 1976) and a t h i g h e r salinitles ( K i t a n o and o t h e r s , 1976). In the sea urchin Arhacia punctulata, r e g e n e r a t e d s p i n e s c o n t a i n e d c a l c i t e r e l a t i v e l y low i n magnesium and ~ i g h i n s t r o n t i u m i n c o m p a r i s o n t o t h e normal s p i n e s . The magnesium c o n t e n t was

132

N. Watabe

directly proportional to the water temperature, which was interpreted to be due to the higher proportions of normal type structure formed in the regenerates at higher temperatures (Davis and others, 1972). The effects of compositions of the external environment on the skeletal CaCO 3 are also complex. A considerable data have been accumulated concerning the in vitro formation of polymorphic species of CaCO 3 in the presence of various Ions at different temperatures (see Kitano and others, 1976 for references). Not all of those results may be directly applicable to the in vivo systems of calcification. As discussed in the section 3, the sites of formation of the crystals in biological systems vary by the skeletal structures and by the organisms, and the composition of the fluid of crystalization is not necessarily similar to that of the external environment. The inorganic composition of the extrapallial fluid is similar to the sea water in marine bivalves, but markedly different from the environmental water in freshwater bivalves (Wada and Fujinuki, 1976). Manganese content in freshwater is not very high, but that in the shell of freshwater bivalves (aragonite)is relatively high (Horlguchi, 1959). Some animals do not deposit CaCO 3 in isotopic equilibrium with their environment. Cnidarians and some algae fractionate oxygen isotopes in their metabolisms and echinoderms show marked fractionation of carbon and oxygen isotopes (see Rye and Sor~ner, 1980). Watabe and Blackwelder (1976) demonstrated that increases of Sr 2+ content by 0.25 - 0.75 mM or ME 2+ by 2.6 - 8.0 ~M in the environmental water caused proportional increases of aragonite in the regenerated shells of Pomacea paludosa. This again is attributed to the accelerated rate of r e c o v ~ o ' ~ a n i m a l s in those concentrations to form normal shell structure (Watabe, 1981). The few examples shown above clearly indicate that the environmental parameters influence the biological CaCO 3 crystal formation in many different ways. Rosenbel (1980) cautioned a generalization of the relationship between the environmental factors and the minor element composition of mollusc shells based on the various data published in the past. Direct comparison of the data is frequently unjustified because of the difference in the age and habitat of the animals, and the portions of the calcareous structures analyzed. It is hoped that systematic and well-deslgned experimental studies will be carried out to understand the mechanisms of environmental control on biological calcification.

ACKNOWLEDGEMENTS

I would like to thank Roni J. Kingsley who has given permission to use her unpublished electron micrographs, to Dana G. Dunkelberger for his assistance in making the photographs, and to Carm A. Finneran for typing the manuscript.

Crystal Growth of Calcium Carbonate in the Invertebrates

133

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Crystal Growth of Calcium Carbonate in the Invertebrates

THE AUTHOR

Dr. Nodmitgu Watabe Born on November 29, 1922 in Japan Received M.S. in Mineralogy in 1948; D.Sc. in Biocrystallography in 1960 from Tohoku University, Japan Research Investigator, Fuji Pearl Col., Japan 1948-1952 Assistant, Faculty of Fisheries, Prefectural Unlveristy of Mie, Japan 1952-1955; Lecturer 1955-1959 Research Associate, Duke University 1957-1970 Associate Professor of Biology, University of South Carolina 1970-1972; Professor of Biology 1972-present; Director of Electron Microscopy Center, 1970 to present R e c e i v e d Elmer W. E l l s w o r t h Award i n P e a r l R e s e a r c h 1952; A l e x a n d e r yon Humboldt Award ( F e d e r a l Rep. Germany) 1976.

147