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Re fe re nc e: B ioL Bu ll . 1 61 : 4 02 â€ ”41. (De cembe r, 1 981)

CRYSTALLINE AXES OF THE SPINE AND TEST OF THE SEA

U RCHIN STRONGYLOCENTROTUS PURPURATUS: DETERM INATIO NBY CRYSTAL ETCHING AND DECORATION

KAYO OKAZAKI,t RICHARD M. DILLAMAN,* AND KARL M. WILBUR

Dep artm en t o f Z oo lo gy , D uk e U niv ers ity , D urh am , N . C . 2 77 06

ABSTRACT

By means of crystal etching and scanning electron m icroscopy the c- and a-axes of skeletal units of Strongylocentrotus purpuratus have been determ ined.Crystal etching and crystal decoration were found to be equally useful in demonstrating that the spines are single crystals of m agnesian calcite. A ll regions ofspine cross sections had common c- and a-axes. C om parisons of etched figures todecorated crystals on the sam e cross section of a spine showed that the grooves of

the etch figures were parallel to the edges of the upper faces of the decoratedc ry sta ls. T he a -a xe s o f th e p rim ary sp in es d iffe re d from th ose o f th e in te ramb ula cra lplates to w hich they w ere attac hed.

C ertain regions of the tubercle w ere show n by SEM exam ination of decoratedtests to be polycrystalline aggregates lacking uniform crystal orientation, w hereasthe rem aining portions of the interam bulacral plates shared the sam e a-axes. Theprim ary plates and dem iplates of the am bulacral plates w ere each single crystalswhose a-axis, and often c-axis, orientations were independent of adjacent compartments.

R efle ctiv e p ro pe rtie s o f d ec ora te d p la te s c oa te d w ith g old -p alla dium h av e b ee nshow n to be useful in dem onstrating differences in crystal orientation betweenadjacent interam bulacral plates and betw een com partm ents of individual am bulac ralpla tes .

INTRODUCTIONThe skeleton of the Echinoidea is of crystallographic interest because of its

ultrastructurend theorie ntationf itsunits.he spinesand platesarem agnesiancalcite form ed as a m ineral m eshw ork of sm ooth trabeculae by cells of the derm isw ithin the interconnecting spaces (W eber et al., 1969; H eatfield, 1971; MÃ¤rkel,1 981). X -ray diffra ction and polarized light studies have indica ted that th e spine sand plates, w ith the exception of tubercles w hich bear spines, are single crystals(Schm idt, 1930, 1932; W est, 1937; D onnay, 1956; R aup, 1962, 1966; C urrey andN ichols, 1967; D onnay and Paw son, 1969; N issen, 1969).

By means of polarization microscopy, it has been shown that in spines the caxis is parallel to the axis of elongation (Schm idt, 1930, 1932; W est, 1937; R aup,1962, 1966) and that in the am bulacral and interam bulacral plates of Strongylocentrotus purpuratus the c-axis is norm al to the outer surface, an orientationcommon to many, but not all, species (Raup, 1962). Other crystal axes were notdeterminedin thesestudiessincethe opticalmethod is limitedto definingthe

Rec eiv ed 3 Aug us t, 1 98 1; a cc ep te d 1 4 S ep temb er , 1 98 1.t Hargitt Fellow, Department of Zoology, Duke University. Present address: Dept. of Biology,

Tok yo Me tr op oli ta n Uni v., T ok yo 1 58 , J ap an .* Present address: Institute of Marine Biomedical Research, Univ. of N. Carolina at Wilmington,

Wilm in gto n, N . C . 2 840 3.

40 2



CRYSTAL ORIENTATION IN SEA U RCHIN 403

direction of the c-axis. X-ray diffraction, which can resolve the c-axis and the a-axes, has been applied to the analysis of echinoid skeletons in only a lim ited wayand has been directed prim arily to the question as to w hether the spines and plates

constitute single crystals (G arrido and B lanco, 1947; D onnay, 1956; D onnay andPaw son, 1 96 9).T he m etho d of crystal deco ration has p roved valuable in the crystallographic

analysis of larval and adult echinoid skeletons (O kazaki and InouÃ© ,1976; Okazakiet al., I 980; D illam an and H art, 198 1 ) as w ell as calcareo us sponge spicules (Jones,1955) and the test of a foraminiferan (Towe, 1977). In this method, skeletal structures placed in a saturated solution of CaCO 3 becom e decorated with calcite crystalsw hose orientation indicates the orientation of the crystals on which they form .O bservations of the decorated crystals by electron m icroscopy perm its determ ination of the c- and a-axes (Dillaman and Hart, 1981).

T he view that the plates of the echinoderm test are single crystals has not founduniversal acceptance. B ased on evidence from polarized light and observation ofsu rface replicas by transm ission electron m icroscop y, T ow e (1 9 67 ) suggested thatinteram bulacral plates, w hich ap pear to be sin gle crystals, a re form ed by oriented

polycrystalline growth followed by a maturation involving recrystallization andc ontinuous fusion and c oalescence. T he evidence presented does not in clude information On the a-axes of the skeletal elements and for this reason cannot completelydefine crystal orientation. B y m eans of Laue X -ray diffraction and crystal decoration, D illaman and H art (198 1) w ere able to show that the inte ram bulacral platesconform to the criteria of single crystals as in dicated by c- and a-axis orienta tion.Further, exam ination of the ultrastructure of the fenestrated plates by scanningelectron m icroscopy gave no evidence of discontinuities or a polycrystallinestructure.

In th e present study w e h ave carried out further de tailed an alysis of the skeletalstructure of Strongylocentrotus purpuratus by the m ethod of crystal etching. Itappearedpossibl ehatby producingetchfigureshecrys tallanesof skeletalnitsmight be made evidentand sopermitthedeterminationfc-and a-axesand crystalorientation. This has proved to be the case. Results obtained by crystal etching

w ere found to give results equivalent to those by crystal decoration. Then, usingdecoration, w e com pared crystal orientation of plates and attached spines and theorientation of the crystal com partm ents w ithin sin gle am bulacral pla tes of the test.

MATER IA LS A ND METHODS

S pecim ens of adult S tron gylocentro tus pu rpuratus obtaine d from P acific B iom arine L aboratories, V enice, C alifornia w ere held in large recirculating sea w atertanks at room tem perature. S am ples of prim ary spines, am bulacral and interambulacral plates w ere prepared by rem ovin g A ristotle's lan tern and in ternal organsfrom living urchins and immersing the rem aining skeletal m aterial and attacheds of t t i ss ue i n C lo ro x ( 5. 25 % s od iu m h yp oc hl or it e) fo r 5 h ou rs . T hi s t re at me nt

rem oved all organic m aterial covering and connecting the skeletal com ponents.A fter 5 w ashes w ith distilled w ater adjusted to pH 8.0 w ith N aO H, prim ary spines

and coro nal plates w ere dehy drated through 70% , 95% and 1 00% e thanol and storedin 1 00% e th an ol.

Etching of spines and plates

For e tc hin g o f sk ele ta l e leme nts, C lo ro x-tre ate d sp in es a nd p la te s w ere re tu rn edto distilled water, pH 8.0, by hydration through 95% and 70% ethanol. Entire



404 OKAZAKI,DILLAMAN,AND WILBUR

spines and those fractured at right angles to the long axis w ere then im mersed in1â€”3%cet ic acid ( pH 2 .6â€”2 .8 ) or 4â€”10minu te s. Co rona l p la te s were d is ar ti cu la teda nd tre ate d w ith 0 . 1 â€ ”0 .0 1%a ce tic a cid (pH 3 .2 â€ ”3 .7 )fo r 1 0 m in ute s. E tc hin g w as

stopped by transfer to 0.01 N NaOH, followed by repeated washes in distilledwater, pH 8.0.

Deco ration of spines and p lates

Etched spines and plates were decorated with calcite crystals following them ethod of O kazaki et al. (1980). Sam ples in distilled w ater w ere transferred intoa 0. 1 M NaHCO3 solution to which was added a 0. 1 M CaCl2 solution to give aratio of the two solutions of 5:2. After 5 m inutes in the final m ixture, specim ensw ere washed with distilled water, pH 8.0, and dehydrated through an ascendings erie s o f e th an ol.

S ca nn in g e le ct ro n m ic ro sc op y

Untre ate d, e tc he d, a nd e tc he d a nd d ec ora te d sp in es a nd p la te s a fte r d eh yd ra tio nw ith ethanol were air-dried and attached to alum inum stubs with Duco cem ent.A fte r sp utte r-c oa tin g w ith g old -p alla dium, sp ec im en s w ere e xamin ed w ith a P hilip s501 scanning electron m icroscope at 15 or 30 KV.

RESULTS

1. Structure of untreated coronal plates and spines.

Coro na l p la te s. T he c oro na c on sists o f 2 0 c urv ed rows o f p la te s, fiv e amb ula cra lareas of tw o-plate row s alternatin g w ith five interam bulacral tw o-plate row s (F ig .lA). Each plate bears one large primary tubercle on which the primary spine issitu ate d. T he re a re se ve ra l se co nd ary tu be rc le s w ith sp in es su rro un din g th e p rim arytubercle (F igs. lD , 6A , B ). A ll tuberc les consist of a b asal po rtion (the boss) havingthe shape of a low truncated cone which is surmounted by a term inal knob (the

m am elon) lying on a narrow shoulder. The boss is encircled by a bare area to w hichare attached the m uscles operating the spine. These four parts of the tubercle aredesignated as regions 1, 2, 3, and 4 (Fig. 6C).

Each am bulacral plate is an assem bly of seven com partm ents, each pierced byse ve n p aire d p ore s fo r th e p assa ge o f th e p od ia (H yman , 1 95 5). T he se c ompa rtm en tsconsist of tw o prim ary plates (Fig. 6A , a, g) and five dem iplates inserted betw eenthem (Fig. 6A , b, c, d , e, f). E very part of the coronal plates exhibits a fenestratedstructure w ith the exception of the outer surface of the m am elon, which is solid(Figs. 6A , B, and C). Under the scanning electron microscope, surfaces of thecoronal plates and spines appear sm ooth and do not display any surface texturesuggestive of th eir crystalline com position, as pointed o ut by C urrey an d N ichols(1967) and Dillam an and H art (1981).

Prim ary spines. The spines of Strongylocentrotus purpuratus have been described in detail by H eatfield (1971). T he m ain regions are a basal portion, a m illed

ring, and a tapering shaft (Fig. 1B, C). The concave surface of the base and themamelon of the tubercle of the associate plate form a ball-and-socket joint(F ig. 1C ).

In tra nsverse se ction, the spine is charac terized by a series of calcite ring s (F ig.2A ) in a continu ous m eshw ork . In the outer zone, w ed ges of so lid calcite alternatewith the m eshwork to form radii and concentric rings. The outerm ost surfaces of



C RYSTAL ORIENTATION IN SEA URCHIN 405

F IG UR E 1. A . Light m ic rograph of C lorox-treated test show ing curved row s of plates, X 2. B . Lightm ic ro gra ph o f s pin e s ho win g th e b as al p ortio n, m ille d rin g a nd ta pe re d s ha ft, X 5. C . S ca nn in g e le ctro n

m icrograph of spine showing concave base, X25. D . Light m icrograph of test show ing double row ofam bulacral plates, each pierced by seven pairs of pores. A ls o apparent are the m am elons that acceptth e s pin e, X 1 0. E . S ca nn in g e le ct ro n m ic ro gr ap h o f b as e o f s pin e s howin g t ra be cu la r c ompo ne nt s b etw ee nth e lo ng it ud in al c olumn s, X 2l0 .

the wedges are smooth and rounded and appear as longitudinal columns in sideview (Fig. lC , E). The number of columns on a single mature spine ranged from40 to 60, and the number frequently increased from the milled ring to the tip ofa spine. In transverse section, the increase was seen as new radii (Fig. 2A). In thepresent study, the wedges forming the columns were loci for observations of etchfigures and decorated crystals.

2. Etch figures and decorated crystals on the spine.

Etch figures created by treating spines with dilute acid are shown in Figures2B, C, D . Figure 2B is a lateral view showing identical cleavage directions on thetwo columns when viewed from the same perspective. Figures 2C, D show etchfigures of two wedges on a cross section of a spine located in regions I and II,respectively, of Figure 2A. On each wedge, three cleavage faces are evident. Thefaces are parallel in the two wedges even though they occupy different positions

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o f rin gs . A ls o n ote n ew ra diu s (R ) th at s erv es to in cre as e th e c irc um fe re nc e, X 4l. B . L on gitu din alco lumn a fter e tch ing w ith acet ic ac id . No te iden ti ca l c leavaged ir ec ti ons in ad jacen tco lumns ,X350 . C .and D . E tch patterns on surface of trans verse section from regions I and II of Fig. 2A. N ote that cleavageangles are identical, X 1250. E. and F. D ecorated crystals on surface of transverse section at sitescom parable to I and II in Fig. 2A. N ote that crystal edges are paralle l, X2500.

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40 7R YS TA L ORIE NTA TIO N IN S EA URCHIN

on the circumference of the spine. Each wedge of the spine shows the same onentation. Since each of the three angles form ed by the intersection of the threecleavage faces w ere 120Â °it w as judged that the observations w ere m ade parallel

to the electron beam along the c-axis of the spine. One can accordingly concludefrom the correspondence of the angles that the spine is a single crystal whose caxis is norm al to the cross-section, that is, parallel to the axis of elongation of thespine. Further, since the angle between the a-axis and the ridge of calcite is 30Â°when viewed along the c-axis (Fig. 3), etch figures can also be used to determ inethe a-axes. From etch figures in Figures 2C, D, the three a-axes of Figure 2A weredeterm ined and are indicated by the superim posed dark lines of Figure 3.

B ecause the etch figures w ere relatively sm all, it w as necessary to m ake m anym easurem ents from a single m icrograph for exact determ inations of cleavage surfaces. By decorating the etched surfaces, determ inations of c- and a-axes wereg re atly fa cilita te d. T he rh ombo he dra l d ec ora te d c alc ite c ry sta ls w ere o f su ffic ie ntsize a nd the coigns p rom inent to the exte nt that the stage of the electron m icroscopecould be oriented so that the ridges of single crysta ls form ed 120Â °angles w ith oneanother. Thus, one could assum e that the crystal was being observed along its c

axis. The a-axes could then be calculated. Figures 2E, F are representative of thehabits of the crystals as observed at all locations on the cross section of a singlespine, includ ing its base. T his result supports the conclusion based on etch figuresthat the spine is a single crystal. A lthough calcite crystals could be grown onu ne tc he d su rfa ce s o f th e sp in e, c ry sta ls o f v ary in g o rie nta tio n fre qu en tly d ev elo pe d,presum ably from sm all chips produced by cutting w hich served as nuclei.

T o test the re lationship betw een etch figu res and decorated crystals, ind ividualspines were first etched and half of the spine then decorated, perm itting a comparison of etch and decorated figures on the sam e spine (Fig. 4A, B). An examin atio n o f th ese fig ure s sh ow s th at th e g ro ov es o f th e e tc h fig ure s p re cis ely p ara lle le dthe ridges of the decorated crystals. H ow ever, as F igure 3 indicates, the ridges on

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F IGURE 3 . Schemat ic rep resent at ion o f cal ci te rhombohedron v iewed a long c -axi s. Bo ld l ines repr es en t th e upper e dges o f t he r homb wh il e dotte d l in es i nd ic ate l ow er e dges .



40 8 OKAZAKI, D ILLAM AN, AND W ILBUR

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FIG UR E 4. Transverse section of a single sp in e view ed with the scannin g electron m icroscope after

etching (A ) and after etching and decoration (B). N ote that grooves of etch figures paralle l the edgeso f the deco ra tedc rys ta ls . A . , X 11,000 , B . , X5000 .

th e fro nt fo rm a ng le s o f 6 0Â °w ith th e rid ge s o n th e re ar o f th e c alc ite rh ombo he dro nwhen viewed along the c-axis (cf. solid lines and dotted lines). Since one wouldinitially assume that the etch figure would represent the removal of calcite alongcleavage faces, it w as th erefore n ecessary to provide an alternative e xplanation forthe relationship between the etch and decorated figures. As an approach to a possibleexplana tion, m ineral c alcite w hose initial cry stal habit w as evid ent w as treated inexactly the sam e m anner as the spin e an d then exam ined w ith the sc anning e lectronm icroscop e. W hen the specim en w as o bserved along its c-axis a s determ ined by itspreexisting cleavage plane (Fig. 5A ), tw o types of etch figures w ere observed, asshown diagrammatically in Figures SB and C. Scanning electron micrographs ofactual pits are shown in Figures SE and F. Figures SB and E correspond to adepression due to the removal of a rhombohedral sector, which was characterized

by sharp grooves and sm ooth etch pit w alls. Equally frequent w ere etch figuresshow n in F igures S C and F w ith groove s that paralleled the ridges of the underlyin gcrystal (Fig. SA). The two lateral walls showed a stepped appearance (Fig. SF)whereas the third wall was smooth. As a consequence, the groove formed wherethe two stepped walls met was not as sharp as in the previously mentioned typeof etch figure. In both types, how ever, the grooves form ed 120Â °angles w ith oneanother when viewed along the c-axis, demonstrating they were equally capableof revealing the c-axis of the crystal. Irrespective of underlying etch figures, orientations of the edges of the decorated crystals were all the same as the initialcalcite (Fig. SD).

3. D ec orated interam bulacral and am bulacral plates.

Interam bulacral plates. E tched and decora ted interam bulacral plate s (F ig . 6B )

exam ined by scanning electron m icroscopy show ed several patterns of the decoratedcrystals. On the tip of the tubercle (Fig. 6C, region 1) the crystals were arrangedin sectors of varying orientation (Fig. 6E). The individual sectors varied in bothth eir c - a nd a -a xe s a nd c on se qu en tly re pre se nte d c oa rse p oly cry sta llin e a gg re ga te s.R egion 2 of th e tub ercle (F ig. 6C ) w as also m ade up of p olycrystalline aggreg ates.This region of the skeleton was less dense than region 1 due to its more open



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FIGURE 5. Schem atic representation of m ineral calcite etched and decorated. C leavage face of

native crystal (A ) and decorated crystal (D ) can be com pared w ith etch pits w ith sm ooth sides (B ) ands teppeds ides (C ) . E . and F . a re scann inge lec tr on microg raphso f e tch p it s such as ind ica ted in B . andC ., re sp ec tiv ely . E ., X l9 00 , F ., X 27 50 .

trabecular structure (Fig. 6F). Crystal sectors in region 2 also showed varying cand a-axes. In regions 3 and 4 of the tubercle (Fig. 6C) and remaining parts ofthe plate, the decorated crystals were especially large at the growing tips of thetrabeculae, and had ridges which were clearly parallel (Fig. 6D ). O rientation of

the sample so as to form 120Â°with all the ridges made it possible to determinethat the c-axis was approxim ately perpendicular to the surface of the plate andfurther perm itted calculation of the a-axes. The relationship between the platemargins (exclusive of the margin with the ambulacral plates) was examined. Thedifference in angle between the a-axes and the sides ranged from 5.9Â°to 9.5Â°witha m ean value for 24 observations of 8.1Â°Â ±5.0Â °.The a-axis directions w ere found

CRYSTAL ORIENTATION IN SEA URCHIN



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412 OKAZAKI,DILLAMAN,AND WILBUR

TABLE I

A shiftof thea-axisforcompartmentc couldnotbe measuredn that thethreeanglesof referencewere not 120Â°.+ , c lockwise.â€”¿ounterclockwise.

adjacent com partm ents w ere view ed w ith only X and Y translation of the sam ple.In sample 1 of Table I, no other compartments shared the same c-axis with compartment e, and their deviation was quite varied. In sample 2, six of the sevencom partm ents shared the sam e c-axis, com partm ent c being the only one show inga difference. However, when the a-axes of the six com partm ents of this sam plewere calculated, it was found that no two had the same orientation. The a-axesranged from â€”¿40Â°o +20Â° relative to that of compartment e (Table I, bottomline). C learly, each com partm ent of the am bulacral plate w as a single crystal w ithcrystal orientation (c- and a-axes) indepe ndent of its adjacent com partm ent.

4 . L igh t r efle ctiv e p rope rtie s o f decor ated and coa ted ske le ta l e lemen ts .

T he thin co at of go ld-palladium o n the surfaces of decorated spin es and coronalplates serv ing a s a condu ctive layer for scannin g elec tron m icrosco py effective lym ade front-surfac e m irrors o f the oute r facets (01 1 1, 1 101 , and 101 1 ) of th e calcite

crystals. The result w as that parallel facets w ithin a given region acted as a singlereflecting surface. Figure 8A shows the upper half of a test which was etched,decorated, coated w ith gold-palladium and illum inated w ith tw o flood lam ps app ro xim ate ly 4 5Â °o th e te st. C erta in in te ramb ula cra l p la te s a pp ea r v ery b rig ht w hileo th ers a re d ark . T he in div id ua l a re as o f amb ula cra l p la te s c on sistin g o f tw o p rim aryand fiv e d em ip la te s a ls o exh ib it ed d iffe re nc es in th eir re fle ctiv e p ro pe rtie s. In F ig ure8B, center, two rows of ambulacral plates are shown. In the left row , the top three

plates do not reflect, and, in the last one, a single dem iplate is bright. In the rightrow , a variety of reflective patterns is seen. The prim ary plates, second from top,are bright, and the dem iplates are dark. The rem aining two plates in the row havea m ixed pattern w ith individual com partm ents varying from very bright throughan interm ediate series of grays to dark. C learly, these results dem onstrate that boththe interam bula cral and am bulacral plates m ay d iffer from adjoin ing plates in theirc ry sta l o rie nta tio n. W ith in in div id ua l amb ula cra l p la te s, d iffe re nc es in o rie nta tio n

between compartments were also evident, as analysis of decorated crystalsh ad sh ow n.

DISCUSSION

T he m eth ods of etching an d crystal decoration co upled w ith scann ing electronm icro scopy u sed in the present stu dy hav e provided in form ation about underlyin g



CRYSTAL ORIENTATION IN SEA URCHIN 413

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crystal faces and their relation to the macroscopic and ultrastructure of skeletalunits of Strongylocentrotus purpuratus. D ecoration of etched spines follow ed byexamination with scanning electron microscopy facilitated the determination of a-axes in that the angles of the coigns of the decorated crystals were distinct andm ore easily m easured than etch figures. In g eneral, how ever, results from etch ingmust be given more weight in that they represent the native crystal faces ratherthan a derivative. E tching and crystal decoration both showed the spines to besingle crystals in that all regions of their cross sections and fine structure hadcommon c- and a-axes.

When etch figures and decorated crystals w ere com pared on the sam e spine,the grooves of the etch figures and the crystal ridges were parallel. Further, identicaltreatm ent of large crystals of m ineral calcite show ed that decorated crystals reliably

reflect c- and a-axes. E tching of these crystals w ithout subsequent decorations gavetwo types of etch figures, one which could be attributed to removal of unit cellsand another parallel to the crystal margins and exhibiting stepping seen in â€œ¿hoppercrystalsâ€• (B uerger, 1970). O ne explanation of â€œ ¿hoppe rr ys ta ls â€ • p ropo se s th at th estepping results from a change in the chemical environment of the crystal as itgrows (Buerger, 1970). W ithin the internal environment of the spines, the magnesium and organic material, including pigment, could conceivably change as aresult of the activity of the mineralizing cells and so give local changes w ithin thecrystal structure. W ith preferential etching of loci differing in lattice structure,such a stepped figure might be formed. Whatever the reason, it is of interest thatmineral calcite with its lower magnesium content and absence of organic materialformed two types of etch figures whereas only the stepped pattern was formed inthe spines. R egardless of the type of etch figure encountered, the observation thatdecorated crystals reflect the same c-axis and a-axes as the underlying crystal as

shown by etching indicates that decoration is a useful method for determiningcrystal orientation of sea urchin skeletal parts.

By etching with dilute acetic acid, it has been possible to confirm the polarizedlight microscopic observations of Raup (1959) showing that the primary spines ofStron gylocentro tus pu rpuratus have their c -axis parallel to the axis of elong ation.Since the a-axes can be determ ined from the three grooves of the etch figures, we



414 OKAZAKI, DILLAMAN, AND WILBUR

could also confirm the conclusion of D onnay and Pawson (1969) from X-ray diffraction that the spine is a single crystal of calcite with a single c-axis and threea-axes.

W e found that the a-axes of interam bulacral plates and their prim ary spinesd id n ot c orre sp on d. One exp la na tio n is th at th e two s tru ctu re s d ev elo p in depend en tlyw ith respect to their m ineralization and w ith control lim ited to their c-axes w hichhave the same orientation. A second explanation is that the base of the spine inrotating on the tubercle tip has a grinding action which breaks off small bits ofthe skeletal m aterial (D onnay and P aw so n, 1969). D uring grow th , th ese b its m ightfuse to a polycrystalline structure as observed by Tow e (1967) and in the presentstudy (Fig. 6E , F).

O ur lim ited an alysis of am bulacral plate com partm ents sho wed w id e diversityof crystal orien tation favoring the view that each com partm ent arises sepa rately.Of 14 plates exam ined (Table I), no two shared the same a-axes. This is noteworthy

in light of the observation that in one plate six of seven compartments had the samec-axis. A na lyses restricted to the c-axes w ou ld ha ve ind icated g reater uniform ityo f c ry sta l o rie nta tio n th an a ctu ally e xis te d.

D iversity of orientation am ong am bulacral plates and com pa rtm ents of sin gleplates w as strikingly evident in w ho le tests that had been etched , decorated, coatedw ith gold-palladium , and then observed w ith directed visible light (Fig. 8 A , B ).Although the sm all m ean size of the crystal faces caused interference betw eenadjacent reflective surfaces w hen view ed with a sm all diam eter beam of visiblelight, this treatm en t w as useful in m aking gen eral assessm ents o f crystal orien tatio ns. D iffe re nc es th at b ec am e e vid en t b y th is sim ple m eth od c ou ld th en b e a na ly ze dp re cise ly fo r c - a nd a -a xis o rie nta tio n u sin g sc an nin g e le ctro n m ic ro sc op y.

ACKNOWLEDGMENTS

We thank Dr. Haskell H art for suggestions concerning the m anuscript. Supported by N ational Science Foundation grant PCM 78-22242.

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1981 okazaki crystalline axes of the spine and test of the sea urchin strongylocentrotus purpuratus...

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