skeletal organizatio inn the coral, pocillopora damicornis by

169

Skeletal organization in the coral, Pocillopora damicornis

By STEPHEN A. WAINWRIGHT(From the Department of Zoology, University of California, Berkeley;present address, Department of Medical Physics, Karolinska Institute,

Stockholm)

With 2 plates (figs. 1 and 5)

SummaryThe skeleton of this Hawaiian reef coral was found to contain at least 99-9% by weightof the mineral aragonite, present as submicroscopic crystals in spheritic arrangements.The organic component of the skeleton comprises o-oi to o-i % of the total weight andhas 3 microscopic constituents: (1) filaments of lime-boring algae, (2) a dispersed net-work of fibres 1 fj. in diameter, and (3) a transparent, milky, regionally birefringentmatrix of chitin. The chitin was observed to be a spongework of fibrils of averagediameter 20 tnfi. The chitin fibrils were inferred to be randomly oriented in the planeof the skeletogenic epithelium perpendicular to the direction of growth of the long axesof the aragonite crystals.

The development of the skeleton is traced from the initial mineral deposit by thelarva after its attachment, through the formation of the larval skeleton and growth intothe fully formed, branching colony. The process of formation of chitin fibrils accord-ing to the contour of the skeletogenic epithelium and the later deposition of aragonitecrystals as described accounts for the formation of all skeletal elements of Pocillopora.

Evidence is presented for the hypotheses that (1) the amide group of the chitinmolecule is responsible for the ability of certain organic substrates to be calcified (thusprotein is not a necessary component of such substrates); (2) zooxanthellae in Pocil-lopora contribute a product of photosynthesis to the coral as the monomer of the chitinmatrix; and (3) chitin synthesis thus depends on the activity of zooxanthellae and therate of chitin synthesis controls the rate of skeletogenesis in Pocillopora.

IntroductionT H E skeleton of scleractinian corals is known to be made up of submicro-scopic to microscopic, acicular mineral crystals which are believed to be arrangedin spheritic aggregations (Ogilvie, 1896; Bryan and Hill, 1941). The mineralis known to be aragonite, the common metastable form of calcium carbonate(Clarke and Wheeler, 1922). The small size of the crystals has preventedany reliable estimate of their dimensions.

Silliman (1846) claimed that 4 to 8% of the skeletal weight of more than 20species of tropical reef Scleractinia was organic, part of which was an ether-soluble wax with a melting-point of 2000 F. Bergmann and Lester (1940)extracted the decalcified skeletons of Acropora cervicornis and Manicina areolatawith acetone and obtained a waxy substance amounting to 0-25% of the totalskeletal weight. After recrystallization, pure cetyl palmate was obtained. Thenon-saponifiable remains were separated into 3 fractions containing (1) sterolsprecipitated by digitonin, (2) non-steroid alcohols (nearly all cetyl alcohol), and

[Quart. J. micr. Sci., Vol. 104, pt. 2, pp. 169-83, 1963.]

170 Wainwright—Skeleton ofPocillopora

(3) hydrocarbons of low melting-point and small amounts of ketones. Theskeleton of M. areolata contained cholesterol.

In his studies of the histochemistry of corals, Goreau (1956, 1959) describeda lamella which is sometimes seen peeling off the external surface of theskeletogenic epithelium in histological preparations of scleractinian tissues.He considered this lamella to be extracellular and therefore part of the skeleton.His histochemical analyses showed this lamella to have the characteristics ofacid mucopolysaccharides. Goreau (1959) suggested a model for the calcifi-cation process in reef corals in which this extracellular acid mucopolysaccha-ride lamella facilitates accumulation of calcium ions at the site of aragonitedeposition and is subsequently active as a template for the deposition ofskeletal mineral.

In Pocillopora damicornis, Wainwright (1962) reported that the majororganic constituent of the skeleton is chitin (polyacetylglucosamine) in an un-usually pure state. The crystalline form of the chitin was not determined, butthe chitin was seen to comprise a network of fibrils of average diameter 20 rmi.The present paper presents data and inferences on the major chemical con-stituents of the skeleton of P. damicornis, their unit shapes and sizes, and theirorientations and physical relationships.

In a penetrating analysis of scleractinian skeletal organization, Bryan andHill (1941) described the aggregations of aragonite crystals throughout theskeletons of Scleractinia in terms of the spherulitic ( = spheritic) crystalliza-tion known to mineralogists. These radial or parallel arrangements of crystalswill form in a gel or magma in a geological situation, in which convectioncurrents are minimal and diffusion is regular. The authors postulate that incoral skeletogenesis the crystals form in an ectodermal mucoid with their longaxes perpendicular to the skeletogenic epithelium. They admit that there is nohistological evidence for such a gel.

Bryan and Hill point out that the vertical skeletal elements (septum, colu-mella) all have trabecular structure of axiolitic aggregates, whereas thehorizontal skeletal elements (tabula, coenosteum) are parallel aggregates pro-duced from a flat surface. The spaces between tabulae express a discontinuousgrowth process as opposed to the continuous formation of solid coenosteum bythe coenosarc. Cessation of growth of a tabula results in the stretching of thetissues attached to the tabula and to the coenosteum. The tissues must detachand then re-attach after they have regained their unstretched shape at a newdistal position. A new tabula is formed in the new position, completely en-closing a cavity, filled with sea-water, in the skeleton.

Materials and methodsLiving colonies of P. damicornis (L.) were collected from the reefs of Porites

compressa around Hawaii Marine Laboratory in Kaneohe Bay, Oahu, Hawaii,in June, July, and August, i960, and March and April, 1961. Skeletons wereprepared for study either after fixation in 10% v/v neutral formalin in sea-water or in Clarke's acetic ethanol, or else without any chemical treatment. In

Wainwright—Skeleton of Pocillopora 171

the latter case, skeletons of unfixed colonies were cleaned of their living tissuesby being sprayed alive, or after being killed and partly decomposed in freshwater for 24 to 48 h., with a jet of fresh water from a high-pressure hose.Cleaned skeletons were then air-dried.

Larval skeltons were obtained from larvae which attached after release asplanulae by colonies maintained in laboratory aquaria. In collections made inthe months of April, June, July, and August, planulae were released in greatestnumbers in aquaria on the nights of the full moon. By the following morning,99% of these planulae had attached to the sides of the aquarium, most of themon the side facing the rising moon, and within 3 in. of the waterline. Theplacing of lucite, cellophane, or loose glass surfaces over the sides of theaquaria permitted the collection of the attached larvae. These were killed atintervals of days, weeks, and months after attachment and were treated in thesame ways as the skeletons of colonies. Some of the most informative observa-tions were made on larval skeletons less than 3 days old. These were examinedalive or after fixation (with neutral formalin solution), dehydration, clearing,and mounting. The specimens were examined by ordinary or 'direct' micro-scopy (Baker, 1956), and. also by phase contrast and with the polarizing micro-scope.

Gross skeletal morphology was studied with a dissecting microscope andcleanliness of the skeleton was assessed by exposing the whole skeleton toaqueous solutions of mercuric bromphenol blue, which demonstrated anyremaining tissue. For studies of the chemical constituents, pieces of skeletonwith any tissue contamination were discarded. Longitudinal, transverse, andtangential sections of terminal, secondary, and basal branches were ground byhand with graded emery papers and carborundum powders and polished onglass plates which had been blasted with graded aluminium oxide. Thesesections were examined by direct and polarized-light microscopy.

Whole colonial skeleton was ground to a fine powder and mounted in aGeneral Electric X-ray diffractometer in which the X-ray diffraction (powder)spectrum was recorded from io° to 650 from the zero order. This informationwas used to identify the mineral component of the skeleton.

For examination of the organic constituents, whole dried or chemicallyfixed skeletal pieces were demineralized in 2% hydrochloric acid or in 10%w/v ethylene dinitrilo-tetra-acetate made up and adjusted with both di- andtetrasodium salts to pH 7-0 to 7-3. The morphology of the organic componentwas observed by direct, phase-contrast, and polarized-light microsopy.

Demineralized skeleton was teased apart with watchmakers' forceps inwater and allowed to dry in air on copper grids covered with formvar. Thesespecimens were shadowed with metal and examined with an RCA EMU 3electron microscope. The following histochemical tests (Casselman, 1959)were made: periodic acid oxidation alone and associated with ninhydrin,alloxan, deamination, methylation, and acetylation treatments all used in con-junction with Longley's (1952) Schiff reagent; alcian blue; azure A and Sudanblack B in 60% tri-ethyl phosphate (specimen extracted with 60% tri-ethyl

172 Wainwright—Skeleton of Pocillopora

phosphate, ethanol, methanol, acetone, or toluene). Other histochemicaltests used were alcoholic and aqueous mercuric bromphenol blue (Mazia andothers, 1953; Bonhag, 1959), toluidine blue (Pearse, i960), sulphuric acid/iodine test for cellulose (Johansen, 1940), Schweitzer's reagent (Fuller andBarshad, i960), and the chitosan-iodine colour test for chitin (Campbell, 1929;Richards, 1951). The decalcified remains of other species of corals were alsosubjected to the chitosan reaction.

Aromatic amino-acids in whole skeleton dissolved in concentrated hydro-chloric acid were sought by analysis of the ultra-violet absorption spectrum(Layne, 1957). The micro-biuret method of Layne (1957) was used todetermine the presence or absence of peptide bonds in whole dissolvedskeleton and in demineralized skeleton.

The relative proportions of organic and inorganic components were deter-mined by weighing clean, dry, whole skeleton before and after demineraliza-tion in acid or EDTA.

ObservationsIn the development of Pocillopora damicornis, the first mineral units of the

skeleton are formed after the planula has attached to the substrate by itsaboral end. Within a day or two of attachment, the tall, glandular aboralattachment epithelium gradually loses its character and becomes the low,squamous skeletogenic epithelium characteristic of the colony. During thisperiod a very thin layer of mineral is deposited in the interseptal areas of thebasal plate. Preliminary X-ray microdiffraction patterns obtained from thismaterial indicate that it is calcite, a form of calcium carbonate not yet recog-nized in extant Scleractinia. The first-order grey interference colour seenbetween crossed polaroids in this structure implies a thickness of 1 to 2 /A.

Within a day or two of attachment, the calcite deposit is covered by thedeposition of spherites of aragonite (identified by X-ray microdiffraction)which are deposited in septal areas delimited by organic deposits of unknowncomposition. These radially arranged crystals grow centripetally and centri-fugally in the development of the spherite. Spherites grow in all directionsuntil they abut on one another and cease to grow in these directions. At thistime, spherites are from 20 to 40 /x in diameter and are piled on one another toform the 6 primary septa of the larval skeleton. The spherites eventually fuseto form trabecular and coenosteal structures of the colonial skeleton, which areaccordingly entirely spheritic in composition; but individual spherites cannotbe distinguished in these.

The colonial skeleton of P. damicornis is dendritic with primary branches 2to 3 mm in diameter and 1 to 4 mm long (fig. 1). Basal branches seldom attaina diameter greater than 1-5 cm, and entire branches may be as much as 10 cmlong. Calices are o-8 mm in diameter, sometimes slightly elliptical, with thelonger diameter oriented parallel to the long axis of the branch. On the

Fig. 1 (plate). Photograph of the skeleton of part of a colony of Pocillopora damicornis.

FIG. I

S. A. WAINWRIGHT


sides of the larger branches calices are distinct and dispersed by one or morecalice diameters, but in the crotches and at the growing tips of the branchesthey are packed so densely that they are polygonal, being separated by coeno-steal walls only o-i mm thick. Septa are either absent or reduced to a columnof spines, which may be longer and finer than coenosteal granulations. Thecolumella, when present, is a low hump in the centre of the tabula that formsthe bottom of the calice.

The skeleton is solid in section except for the spaces between tabulae alongthe growth axes of the corallites (fig. 2, c). Most of the skeletal surface is

FIG. 2. Diagrammatic representation of growth in Pocillopora damicornis asseen in longitudinal sections of branch tips of colonies. A, young branch orcolony; B, result of longitudinal growth of A; C, result of radial growth of B.

coenosteum covered with fine granulations which differ greatly in size, form,and density within a single colony. In ground sections it can be seen that theonly structure at all resembling the typical scleractinian trabecula is the skeletonformed at the growing branch tips (fig. 2, A) where calices are crowded. Onlyin these structures is there a distinct arrangement of radially arranged mineralcrystals. Throughout the coenosteum and tabulae, virtually the entireskeleton of the colony, the mineral crystals appear to be arranged in parallel,though oriented radially with respect to the long axis of the branch. Thus, thesame process of crystal orientation may occur throughout the formation of theentire skeleton. Mineral crystals are seen always to be formed perpendicularlyto the skeletogenic epithelium, in confirmation of the analysis of Bryan andHill (1941). Thus a sharply folded epithelium produces a trabecular structure,whereas a flat epithelium produces the coenosteal and tabular structures withcrystals in parallel.

The diffractometer tracings of the X-ray diffraction spectrum of powderedwhole skeleton show reflections with relative intensities characteristic ofaragonite. No reflections characterizing other mineral or organic constituentsappear in these tracings, and it can therefore be concluded that the mineral ofthe colonial skeleton of this species is aragonite. The method carries a maxi-mum 2% error.

The insoluble organic component remaining after demineralization of whole,


fixed skeleton is transparent, slightly milky, and of the same shape as theskeleton before demineralization. This identity in form exists in the finestmicroscopic detail. When deformed, the hydrated organic component shows avery slight tendency to return to its original shape, and it is easily torn. Whilebeing dried in air, the organic component tends to collapse with the retreatingmeniscus. The dried material is an opaque, brittle mass of a bright white sub-stance.

Gravimetric analysis showed that the organic component amounts to o-oito o-i% by weight of the total dry skeleton. This is thus the most highly

FIG. 3. Camera lucida tracing of a fragment of a thallus ofOstreobium sp. from the skeleton of Porites compressa.

mineralized animal structure known, being nearly an order of magnitude higherin percentage mineral content than the enamel of adult man (Eastoe, i960).

Study of the demineralized skeleton by direct microscopy reveals 3 readilydistinguishable constituents: (1) occasional filaments of lime-boring algae; (2)a loose, dispersed network of fibres about 1 /x in diameter; and (3) a trans-parent matrix that constitutes the bulk of the organic component whose grosscharacters are described above.

Lime-boring algae are to be found in the skeletons of all live and dead coralsthat inhabit the lighted areas of oceans. Although certain Cyanophyta andRhodophyta have been found in this habitat, the most abundant are siphona-ceous and chaetophorous Chlorophyta (Bornet and Flahault,i88o,). Ostreobium(Phyllosiphonaceae) is the genus most often found in the skeletons of Hawaiiancorals. Its physiology is virtually unknown. Weber-van Bosse (1932) found5 species of the genus associated with various reef-dwelling organisms. I havecultured Ostreobium sp. (fig. 3) from Hawaiian Porites compressa. The dis-tinguishing morphological features of as many as 3 of the described specieswere to be found in a single thallus. No specific name will be used in thisaccount.

Removal of the algal filaments from whole or demineralized skeleton withoutdrastic chemical treatment is not feasible. Although such unavoidable con-tamination hindered the assay by chemical micro-analysis of constituents


intrinsic to the coral, the algal filaments did serve as positive controls for manyof the histochemical reactions (table 1; see appendix, p. 183).

Neither the chemical nature nor the distribution of the network of micro-scopic fibres was easy to characterize and they were absent from quite largevolumes of skeleton. For these reasons they have not been considered in theanalysis of organization presented here. It is possible that they are organicremains of an alga or of a fungus, though these interpretations are not sug-gested by their appearance (fig. 4).

The matrix appears completely amorphous by light and phase-contrast

FIG. 4. Camera lucida tracing of microscopic fibres from adecalcified skeleton of Pocillopora damicornis.

microscopy. The extremely diaphanous nature of the matrix was apparentfrom its phase-shifting properties observed under the phase-contrast micro-scope.

Although a number of histochemical tests were performed to elucidate thechemical nature of the matrix (table 1), no reaction of the isolated matrix ap-peared identical to control reactions performed on paraffin or ester-wax sectionsof the coral's soft tissues. The physical nature of the matrix was so dissimilarto that of cellular tissues that comparison of the results of histochemical testson matrix with those on tissues is invalid until such comparison can bedemonstrated empirically to be valid. The X-ray diffraction powder pattern ofthe matrix was shown to be that of chitin (Wainwright, 1962), and the micro-analytical methods used to determine the presence of protein in the skeletongave no indication of the presence of soluble or fibrous protein.

Electron micrographs of demineralized skeleton (fig. 5) showed the matrix asa spongework of fibrils of average diameter 20 m/x. Though these fibrilsshowed no longitudinal periodicity, it is possible that the particles of chromiumand of platinum-palladium alloy used in shadowing may have masked a subtle


periodicity. The only recognizable character was a tendency for fasciation.Though the fibrils appeared oriented in some of the micrographs, theirorientation was such that it could have arisen from the stresses exerted on thematrix during preparation for electron microscopy.

Microscopic examination by polarized light showed the matrix of decalcifiedsections of skeleton to be weakly birefringent in certain regions of the coeno-steum adjacent to the calices. Because the magnitude of this birefringencecould be altered by deformation of the matrix, it was judged to be form bire-fringence. Matrix of the coenosteum seen in sections cut transversely orlongitudinally to the branch axis showed a weak negative birefringence relativeto the direction of coenosteal growth. This direction is perpendicular to thelongitudinal branch axis. In sections cut tangentially to the branch, the matrixof such areas was isotropic.

From these observations with the electron microscope and the polarizingmicroscope, it has been inferred that the fibrils of chitin in the coenostealregions of the skeleton are oriented in the plane of the skeletogenic epithelium,perpendicular to the direction of coenosteal growth and parallel to the longi-tudinal branch axis, and that within that plane the fibrils are randomly oriented.

Pieces from the coralla of several other species of Scleractinia have likewisebeen demineralized and examined microscopically. Species of Pocilloporaalone showed an organic component which maintained its shape and con-sistency throughout demineralization, and which was positive in the chitosan/iodine test. All optical methods were performed on the skeletons of 3 otherspecies of Pocillopora, and the properties of the organic component did notdiffer among the species. In skeletons of genera other than Pocillopora, theonly microscopically distinguishable constituent common to all species wasthe boring alga, Ostreobium. Algal filaments were present in greater numbersper unit volume of skeleton in other genera than in Pocillopora. Dried speci-mens of Seriatopora from the reference collection of the Hawaii MarineLaboratory were used for this examination: all others were collected in thefield by the author. The species examined, their families (Vaughan and Wells,1943), and their sites of collection were the following:

SeriatoporidaePocillopora meandrina, windward Oahu, Hawaii.P. eydouxi, Penguin Spit, Palmyra, Line Is.Pocillopora sp., Penguin Spit, Palmyra, Line Is.Seriatopora hystrix, Eniwetok lagoon, Marshall Is.S. angulata, sea reef, Ine, Arno Atoll, Marshall Is.

Acroporidae

Acropora sp., Penguin spit, Palmyra, Line Is.

FIG. 5 (plate). Low-power electron micrographs of decalcified skeleton of Pocilloporadamicornis. A, specimen shadowed with chromium; B, specimen shadowed with platinum-palladium.

BFIG. 5

S. A. WAINWRIGHT


Mussidae

Lobophyllia sp., Penguin Spit, Palmyra, Line Is.

FaviidaeCyphastrea ocellina, Kaneohe Bay, Oahu, Hawaii.

Fungiidae

Fungia scutaria, Kaneohe Bay, Oahu, Hawaii.

TrochosmiliidaeDendrogyra cylindrus, Salt Gut Reef, Jamaica, W.I.

DendrophylliidaeBalanophyllia elegans, Frenchman's Reef, south Moss Beach, California.

InterpretationAn individual of the species P. damicornis begins life independent of the

parent as a planula with 12 mesenteries, an anthopharynx, and a button ofcolumnar, glandular epithelium covering the aboral end which secretes a stickysubstance. The free-swimming planula does not normally form a mineralskeleton. Within a day or two after attachment, the aboral attachment epithe-lium changes to a thin epithelium in which cell membranes have not yet beenobserved and which is the skeletogenic or calicoblastic epithelium of thecolony. During this period a layer of mineral, probably calcite, 1 to 2 p thickis deposited on the substrate externally to the tissues of the coral in the inter-septal areas of the developing basal plate. After its attachment the larva re-mains on this sticky attachment substance and may or may not secrete a secondorganic substance or alternatively more of the original attachment substance.

Simultaneously with the formation of the calcite patches, the 6 primary septa,which are present only in the larval skeleton, begin to form as groupings ofspherites of aragonite. Each spherite continues to grow in every radial directionuntil it either abuts against its neighbours or until its formation is stopped bythe controlling skeletogenic epithelium. The spherites eventually fuse in theformation of the growing skeleton so that by the time the colony has developed20 or 30 polyps and calices and a considerable amount of coenosarc, theskeleton has attained the 'adult' colonial form, composed of solid coenosteumbetween calices and with tabulae set at more or less regular intervals along thegrowth axes of the corallites in the central core of each branch.

The young colony for a while is merely a cluster of terminal branches.From this stage the condition of the fully formed colony, whose branches ofhigher order show a chambered centre and solid cortex, is attained by growthin length and growth in diameter, the latter two components operatingsimultaneously.

Growth in length is accomplished by the deposition of trabecular terminalcoenosteum which separates apical calices and never attains a thicknessexceeding 0-3 mm. When this thickness is exceeded, the space between


polyps is sufficient for a new polyp to arise by intertentacular budding, and itis only at these growing branch-tips that new polyps arise. The developmentof new polyps accounts for the crowding of polyps at branch tips. Non-growing branch tips are characterized by polyps and calices which areseparated by more than half of their own diameter. In effect, the increase inbranch length is concomitant with asexual reproduction of polyps and thegrowth of their associated skeleton (fig. 2, A, B).

Growth in diameter does not involve the development of new polyps, but isaccomplished by a three-dimensional thickening of coenosteum accompaniedby continued growth by lateral polyps. Since the polyps themselves do notincrease in diameter, the result is a growth in diameter of the branch and anincrease in the distance between calices (fig. 2, c). The continued growth bypolyps in the crotches of branches which are increasing in diameter causes adecrease in coenosteal area, rather than elimination of polyps, and this resultsin crowding of polyps in these regions.

As long as a polyp is at the apex of a growing branch-tip or distant from itby only a few polyp diameters, the coenosteum grows more quickly than dothe tabulae; thus the polyp bases are lifted discontinuously from their tabulae,and new tabulae form in the resultant distal positions. As a polyp is forced tothe side of a branch tip by the intercalation of new polyps at the apex, thegrowth rate of the immediately adjacent coenosteum decreases until it equalsthat of the tabular growth rate. Thus, as the polyps grow laterally to thebranch, tabular growth rate equals coenosteal growth rate and a solid skeletalcortex surrounding a chambered central core is produced (fig. 2, c). Theserelationships have been observed to apply in the 4 species of Pocillopora ex-amined which range in form from the delicate P. damicornis to the sturdy P.eydouxi whose basal branches are as much as 8 cm in diameter. In otherspecies of Pocillopora not yet examined, the formation of spaces and tabulaeoccur throughout the corallum.

The centres of spheritic structures in coral skeletons have been described asbeing dark and isotropic in transmitted light and have been called 'centres ofcalcification' (Ogilvie, 1896). Few authors have ventured a guess at the natureof these centres, though Bryan and Hill (1941) state that they appear to arisefrom the presence of excessively finely divided material interstitial to thecrystals. In the present study, histochemical tests indicate that the organiccomponent of the centres does not differ chemically from that of the peripheryof the spheritic structures.

If, as inferred above, the chitin fibrils of the matrix are oriented randomlyin the plane of the epithelium and perpendicularly to the long axes of thearagonite crystals, there are two possible ways in which this orientation mayarise. Their arrangement may in no way reflect the arrangement or shapes ofthe crystals, that is, they may run through the crystals as well as betweenthem; or as shown in the diagrammatic interpretation (fig. 6), they may runonly between crystals. The latter arrangement could come about in the follow-ing manner. Chitin fibrils may be secreted either in a pattern or at random by


the skeletogenic epithelium, and the advancing front of aragonite crystals,which grow in length faster than in diameter, may push its way into thefibrillar spongework. As crystal growth proceeds, the fibrils would first beforced to orient in the plane perpendicular to the axis of fastest growth of thecrystals. As the crystals grew in diameter, they would force the fibrils into apattern such as that shown in the figure (fig. 6). With crystals whose shortaxes are below the limits of resolution of the light microscope, this arrange-

cell membrone

-oragonite

FIG. 6. Diagrammatic interpretation of the relationshipsbetween chitin and aragonite during skeleton-formation in

Pocillopora.

ment of fibrils between crystals would appear random under the polarizingmicroscope.

It is hoped that examination with the electron microscope of replicas offractured and etched surfaces of coral skeleton will give the informationnecessary to choose between these two alternatives. Meanwhile there is someevidence from electron micrographs of the fibrillar matrix which has beensubjected to an unknown degree of disorientation. In such material, therepeated occurrence of patterns of fibrils such as that presented here (fig. 5,B) suggests that such fibrils have lain in a position determined by the flat faces


of mineral crystals, whose growth along their short axes has forced the fibrilsto conform to the shapes of the crystals.

DiscussionThe bulk of the literature on the organic matrices of mineralized animal

structures is concerned with the nature of the protein components. Indeed, inthe few species for which quantitative data are available for the organic con-stituents of mineralized matrices, except some Crustacea (Lafon, 1948), thereappears to be an overwhelming proportion by weight of protein over lipid andpolysaccharide components. In bone, dentin, and enamel a complex situationinvolving matrices of all classes of compounds has come to be recognized.Proteins, nevertheless, have received most attention hitherto. It has beenshown in bone that apatite crystals form within fibrils of collagen, and researchon this functional relationship is revealing the active role played by organicmatrices in mineralization processes (Glimcher, i960).

However, a case for the functional importance of polysaccharides inmineralization is also being made and the present work strengthens this case.Sobel (1959) describes the experimental evidence that the conditions and theresult of calcification experiments in -vitro with pure reconstituted collagen andwith acid-treated decalcified bone are less like the conditions of calcificationin the living organism than are those of experiments in vitro in which the pro-teinaceous substrates have been treated with chondroitin sulphate, the acidmucopolysaccharide of bone.

Lafon (1943) concluded that the small amounts of protein found in thecalcified cuticles of some marine Crustacea have the function of rendering theorganic cuticle calcifiable, because calcium salts '. . . would not deposit onpure chitin'. Later (Lafon, 1948) he suggested that a high chitin contentrendered the cuticle more calcifiable, though in neither paper is evidencepresented of the effect on calcification of any component of the cuticle. Sim-kiss (i960) tested the metachromatically staining regions of the shells of bi-valves, Anodonta cygnea and Cardium edule, for removal of metallic ions fromsolution. He concluded that chelation was the probable mechanism, the ionbeing held by an acid group. Wilbur and Watabe (1962) have given criticalexperimental evidence that the type of protein deposited in the shell of severalmolluscs corresponds to the type of mineral subsequently deposited on it.

The studies just cited strongly support the hypothesis that the organiccomponents of mineralized animal skeletal elements function in skeletogen-esis. The following mechanisms may be involved. Organic components maycontain the sites of seeding of the mineral crystals; they may facilitate crystalformation by actively concentrating calcium ions by chelation in the region tobe calcified; they may determine the crystal form of the mineral deposited;they may control the shape, size, and orientation of the crystals. These studiesalso suggest that the presence of a protein is required to render the matrixcalcifiable.

The results of the present study are consistent with some of these sug-


gestions, but perhaps sharpen the focus on the significance of the presence ofpolysaccharide in a mineralizing system. This coral seems not to requirefibrous protein as a site for the seeding or growth of aragonite crystals; nor doacid mucopolysaccharides seem to be present in the skeletal matrix. Instead, aneutral amino-polysaccharide, chitin, seems to fulfil all the necessary functionsof an organic matrix in the formation of skeleton. The chitin fibrils may wellhave the physical properties of flexibility and strength which are required in asystem which must continually make spatial adjustments as mineral crystalsgrow through it. Chitin also has a chemical trait in common with protein,namely, the amide group of the chitin and the peptide bond of the protein.The hypothesis that the amide group, or a part of it, renders certain animalstructures calcifiable is therefore put forth for future testing.

It is interesting to consider the situation in which this pure chitin matrixexists. It is known that utilizable nitrogen is one of the substances whosescarcity limits the rates of primary productivity in the warm surface waters oftropical seas. Pocillopora has perhaps been adapted to nitrogen shortage byhaving a skeletal matrix of a material whose nitrogen content is lower thanthat of protein.

In some fungi (Cantino and others, 1957) and in an insect (Candy and Kilby,1962) where the biosynthesis of chitin has been studied, the last steps insynthesis are believed to be a polymerization of acetylglucosamine. In otherwords, a chitin micelle is not made by the addition of side groups to a micelleof cellulose. As the Anthozoa are carnivorous, and corals are not known to beexceptional (Yonge, 1940), it might seem strange that a carbohydrate isavailable in sufficient quantities to act as the sole organic monomer for anextensive skeletal system. However, consideration of the manner of life ofthese corals may give a clue to the source.

Pocillopora is one of the genera of so-called hermatypic corals whosegastrodermal tissue is infected with intracellular dinophycaean symbionts,zooxanthellae, whose role in the symbiosis is currently much discussed. It isknown that Chlamydomonas passes into the culture media monosaccharidesand other small organic compounds (Allen, 1956). It is also known that radio-active carbon can be fixed photosynthetically by zooxanthellae and sub-sequently passed to the cells of its anthozoan host, where it may be in the formof a nutritive substance (Muscatine and Hand, 1958) or a vitamin or growthfactor (Goreau and Goreau, i960). A testable hypothesis based on the infor-mation just given is this: that in Pocillopora a function of zooxanthellae is thecontribution by diffusion or by active transport of glucose, or its derivative,which is incorporated into the skeleton of the coral as the monomer of chitin.

Examination of whole skeleton of Pocillopora before, during, and after decal-cification permits the observation that the organic component of the skeletonpermeates the entire skeleton in all details of structure visible in the lightmicroscope. There is evidence, from the observation of the development ofspherites in the larval skeleton, for the supposition that corals and animalsfrom other phyla produce the organic components of their skeleton before they


produce the mineral components. Most of the known functions of the organiccomponent depend upon this sequence. It follows that the process whoserate controls the rate of skeletogenesis (or shell formation) in nature may betheproduction of a calcifiable organic substrate. Since Goreau (especially 1961)has shown that the activities of zooxanthellae have a potentiating effect onskeleton formation in many species of hermatypic corals, the suggestion is madehere that in Pocillopora this potentiation may indeed be a nutritive one, namely,that of supplying the building blocks of the organic component of the skeleton.

The author is grateful to the Department of Zoology at Berkeley, theHawaii Marine Laboratory, and the Edwin Pauley Grant for facilities andsupport during this work. He also gratefully acknowledges the critical readingof the manuscript as well as many more profound aspects of training of Dr.Cadet Hand and Dr. Sydney Smith. This paper is no. 187 from the HawaiiMarine Laboratory.

ReferencesALLEN, M. B., 1956. Arch. Mikrobiol., 24, 163.BAKER, J. R., 1956. Nature, London, 177, 194.BERGMANN, W., and LESTER, D., 1940. Science, 92, 452.BONHAG, P. F., 1959. Univ. Calif. Pub. Ent., 16, 81.BORNET, E., and FLAHAULT, C , 1889. Bull. Soc. bot. France, 36, p. cxlvii.BRYAN, W. H., and HILL, D., 1941. Proc. roy. Soc. Queensland, 52, 78.CAMPBELL, F. L., 1929. Ann. ent. Soc. Amer., 22, 401.CANDY, D. J., and KILBY, B. A., 1962. J. exp. Biol., 39, 129.CANTINO, E. C , LOVETT, J., and HORENSTEIN, E. A., 1957. Amer. J. Bot., 44, 498.CASSELMAN, W. G. B., 1959. Histochemical technique. London (Methuen).CLARKE, F. W., and WHEELER, W. C, 1922. U.S. geol. Surv. prof. Pap., 124, 1.EASTOE, J. E., i960. Nature, 187, 411.FULLER, M. S., and BARSHAD, I., i960. Amer. J. Bot., 47, 105.GLIMCHER, M. J., i960. In Calcification in biological systems, edited by R. F. Soggnaes.

Washington (A.A.A.S.).GOREAU, T. F., 1956. Thesis. New Haven (Yale University).

1959- Biol. Bull. Wood's Hole, 116, 59.1961. Endeavour, 20, 32.and GOREAU, N. I., i960. Science, 131, 668.

JOHANSEN, D. A., 1940. Plant microtechnique. New York (McGraw-Hill).LAFON, M., 1943. Ann. Sci. nat. Zool. Biol. anim., 5, 113.

1948. Bull, l'lnst. ociSanog., 45, No. 939, 1.LAYNE, E., 1957. lnMethods in enzymology, vol. 3, edited by S. P. Colowick and N. O. Kaplan.

New York (John Wiley).LONGLEY, J. B., 1952. Stain Tech., 27, 161.MAZIA, D., BREWER, P. A., and ALFERT, M., 1953. Biol. Bull. Wood's Hole, 104, 57.MUSCATINE, L., and HAND, C, 1958. Proc. nat. Acad. Sci., 44, 1259.OGILVIE, M. M., 1896. Phil. Trans, roy. Soc. Lond. B, 187, 83.PEARSE, A. G. E., i960. Histochemistry, theoretical and applied. Boston (Little, Brown).RICHARDS, A. G., 1951. The integument of arthropods. Minneapolis (University of Minnesota

Press).SILLIMAN, B., 1846. Amer. J. Sci. Arts, 51, 189.SIMKISS, K., i960. Proc. malac. Soc. Lond., 34, 89.SOBEL, A. E., 1959. Bull. Jewish Hosp., I, 5.VAUGHAN, T. W., and WELLS, J. W., 1943. Geol. Soc. Amer. spec. Paper No. 44.WAINWRIGHT, S. A., 1962. Experientia, 18, 18.WEBER-VAN BOSSE, A., 1932. Me'm. Mus. roy. d'Hist. nat. Belgique, Hors ser., 6, 1.WILBUR, K. M., and WATABE, N., 1962. In press.YONGE, C. M., 1940. Sci. Rep. Gr. Barr. Reef Exped., B.M. (N.H.), 1, 353.

Wainwright—Skeleton of Pocillopora

Appendix

183

TABLE I

Results of histochemical analysis of the decalcified skeleton of P. damicornis

LlPIDSSudan black B (= SBB)

in 60% tri-ethyl phosphateSBB after 24 h. extraction in tri-ethyl

phosphateSBB, ethanol extractionSBB, acetone extractionSBB, methanol extraction

POLYSACCHARIDESperiodic acid / Schiff (= PAS)Schiffesterification/PASesterification/Schiffmethylation/PASmethylation/Schiffdeamination/PASdeamination/Schiffsaliva/PASsaliva/Schiffazure A (metachromasia)

Tests for cellulose:75% H2SO4Schweitzer's Reagent

chitosan-iodine (for chitin)PROTEINS

alloxan, Schiff, aqueousalloxan/Schiff, alcoholicninhydrin/Schiff, aqueousninhydrin/Schiff, alcoholicSchiffmercuric bromphenol blue:

alcoholic (Mazia and others, 1953)aqueous (Mazia and others, 1953)alcoholic (Bonhag, 1959)

Algalfilaments

+ +

++++

+ + +0

+ + ++ +

+ + ++ + ++ +

0

++0

++0

+ ++ + ++ +

+ + +0

+++

Microscopicfibres

+ +0

++0

+ + +0

0

0

+ +0

+ +0

+ +0

0

0

0

0

0

0

0

0

0

0

0

0

Matrix

weak +

weak +weak +weak +very weak +

+0

0

0

0

0

0

0

0

0

0

0

0

+ + +

00

0

0

0

0

00

+ + = strong reaction; + = weaker reaction; o = no reaction.

skeletal organizatio inn the coral, pocillopora damicornis by

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