Vol. 76. No. 7, pp. 3406-3410. JuJy 1979 Evolution

Interior remodeling of the shell by a gastropod mollusc (biomineralization/Coiius/shell dissolution)

A l a n J. K o h n , E l iz a b e t h R. N4y e r s , a n d V. R. M e e n a k s h i

Department of Zoology, University of Washington, Seattle, Washington 98195

Communicated by W. T. Edmondson, April 26.1979 t ' a b s t r a c t As the Ckmus shell grows by spiraling of the outer lip around the axis, profound internal shell dissolution Ains the walls of the protected penultimate whorl from several millimeters to <50^tm. Shell material is added to the inside of the spire and the anterior part of the columella. The resulting shell has a uniformly thick last whorl and thickened spire that enhance defense against crushing predators and a greatly ex­ panded interior living space for the animal.

The molluscan shell has gained prominence in recent years as an especially favorable system for the analysis of biominerali­ zation processes (1^). Much less attention has been paid to shell dissolution, a co.ntinuing, permanent, and profound process that alters exterior and interior surfaces of the shell in certain pro- sobranch gastropods (5-7). In the genus Conus, dissolution of the internal walls of the shell is particularly striking while shell material is added from within to thicken regions of the shell some distance from its growing edge. Although these renova­ tions have not been studied previously, the resulting very thin inner wall structure has long been known (8) and was used as the primary character separating subfamilies of the Conidae in an early classification (9). In this study we addressed the following questions: (i) What regions and layers of the shell are involved in dissolution and Fig. 1. Axial section of shell of C. liuidua Hwass in Brugu thickening? (it) Of the shell material deposited by the animal illustrating terms used. The outer lip is at right (also the animal's r during its life, how much is later dissolved? (iii) How much of side); the anterior end is at the bottom. Outermost calcified la> the animal’s living space within the shell does dissolution pro­ appears whit« but contains a yellow pigment in C. lividua. Lay , duce? (iv) What is the adaptive significance of interior shell contains dark purple t« brown pigment visible in the photogr4 rertiodeling? Inner layers 3 and 4 appear white and indistinguishable from ( other. Lines mark percentages of total aperture height and corresf to cross sections in Figs. 2 and 3. i, Posterior region of whorl wi MATERIALS AND METHODS dissolution does not occur; ii, depositional region at adhesion ; on previous whorl; iii, depositional region on abapical surface of We selected Conus lividus Hwass in Bruguiere, an inhabitant solved previous whorl; col, anterior thickening of columella; ri of tropical Indo-West Pacific coral reefs, as representative of spiral ridge in last whorl. the genus. Shells collected in Hawaii were filled with resin to preserve the integrity of the inner whorls and then were sec­ tioned axially or transversely to permit measurements of shell the several layers comprising the gastropod shell (11). ^ thickness. Etched, polished, gold-coated sections were used to mantle on the animal’s right side secretes the growing edg aid visualization of boundaries between shell layers. For scan­ outer lip of the dextrally coiled shell. The outermost shell Ie; ning electron microscopy, pieces of fractured shell were cleaned is the uncalcified proteinaceous periostracum. Inward of in dilute sodium hypochlorite and coated with gold or gold/ are several layers of crystalline CaCOs with a glycopro palladium. matrix of 2% or less by weight (1, 12). Primitive prosobra gastropods (order Archaeogastropoda) vary widely in or; RESULTS form and architecture (13), Shells of the most derived o Neogastropoda are structurally more uniform, consisting In C. lividm as well as most species in the genus, both spire and marily of three or four aragonitic layers of linear or brand last whorl are conic; thus, the whole shell appears biconic, with crossed lamellar crystal architecture (14, 15). This consis' ' a long, narrow aperture (Fig. 1). As the shell grows, its shape series of elongate, curved, branching and interdigitating remains constant (10). mary lamels, each comprised of secondary lamels oriente To appreciate dissolution, one must understand how the shell parallel laths. Each secondary lamel consists of tertiary Ian is produced. Histochemically differentiated regions of outer these are parallel needle- or rod-like crystals (Fig. (13- mantle epithelium and underlying calcium gland cells secrete 2E) The secondary lamels in adjacent primary lamels are orie/ at a characteristic angle [commonly, 82° (14); mean ± SD, 8' ■ The publication costs of this article were defrayed in part by ^ g e charge payment. This article must therefore be hereby mark^ "ad­ ± 5.5° in layers 1 and 3 of a specimen of C. lividus]. This vertisement” in accordance with 18 U. S. C. §1734 solely to indicate strong, interwoven or interlocking structure analogous to this fact. wood (16, 17). Although the pattern is not rigidly mainta Thickness of the shell, and of each component layer, is rather uniform proximal to the growth region of the outer lip in the posterior region of the last whorl (Figs. 2B, 3 A, B, E, and F). Layer 2 is the thickest (Fig. 2E) and strongest; it determines the path of shell breakage, particularly near the outer lip where the gro\ving shell is thin and most susceptible to damage from ac­ cident or attempted predation (18), The shell is thickened in two ways anterior to the midregion of the last whorl, primarily by layer 3-. (i) between about 225“ and the columella (Fig. 1, col; Fig. 2 C and D; major peaks in Fig. 3C; high end points of curves in Fig. 3D), and (ii) a spiral ridge characteristic of C. lividus and certain other species of Conus but not of general occurrence in the genus (Figs. 1 and 2C; peaks in Fig. 3D and at left in Fig. 3C). Figs, 2B and 4 show that the shell does not merely gradually thicken as it grows. The walls of the penultimate and earlier whorls are extremely thin, due to dissolution that begins slightly more than one revolution in from the outer lip (range 372“- 456°; mean, 413°; n = 13) and is mostly completed within an arcof 85°-160° (mean, 112°; n = 8). If thinning of the penul­ timate whorl did not keep pace with thickening of the last whorl, the narrow aperture of the shell would be nearly oc­ cluded (Fig, 2B), Etched, polished sections and scanning electron micrographs of fractured shells in the region of dissolution show that shell material is dissolved smoothly and without regard to crystal architecture (Fig. 4C). We assume that the mantle on the ani­ mal’s left side is applied to the shell and causes its dissolution in this region, but we base this on anatomy of the animal re­ moved from its shell rather than on direct observation. Layer 1 is dissolved first, at about 390°-400° at the 80% level (Figs. 2B and 3£) and further inward (420°-470°) anteriorly (Figs, 3 and 4A). Thinning of layer 2 begins immediately at the point of its exposure to the mantle; it completely disappears at IG. 2. C. lividus. (v4) Cross section of outer lip, showing 450°-495° (Figs. 3 and 4 B and C). Layer 3 then immediately . sed-lamellar crj’stal microstructure of layer 1 at growing edge (left) begins to thin (Fig. 4C). Generally it does not completely dis­ layer 2 beginning on inner depositionaJ surface (arrow). (Light appear, but some regions of inner whorls in the posterior third rograpli of etched, polished, gold-coated specimen; scale bar = consist only of layer 4 (Fig. 3£). The innermost walls, consisting mm.) (B) Cross section at 80% level (Fig. 1), showing system of eating position by degrees from outer lip and regions of thickening of layer 3 or 4 or both, remain patent throughout (Fig. 4D; gaps -90°) and dissolution (380°-480‘’). (C) Cross section at 32% level, in Fig. 2B are artifacts of preparation), but they are extremely OTng spiral ridge and thickening of central columellar region, thin, often 35-50 (im (Figs, 1 and 2B). They presumably narily by layer 3. (D) Cross section at 20% level, showing columellar function to support the digestive gland, which fills all early kening. {B-D, etched, polished, uncoated specimens; scale bars whorl space within the spire. mm.) (£) Cross section at 50% level, about 90° from outer lip, Mng lamel structure and orientation in layers 1, 2, and 3. Outer As the Conus shell grows by helical progression of the outer I surface is at top; inner or depositional surface is at bottom right. lip around the axis of coiling, the new growth covers all but the ( idth of primary lamel; 2', arrow indicates secondary lamels within most posterior part of the previous whorl. This results in a spire lary lamel; 3', arrows indicate edges of tertiary lamels on surface that, unlike the last whorl, is not covered by subsequent external rimary lamel. (Scanning electron micrograph of fractured, gold- deposition of shell, but remains exposed to attack by predators, ted specimen; scale bar = 0.05 mm.) boring organisms, and physical environmental stresses for the animal’s entire life. ause the primary lamels curve, the long axes of primary The thick shell in the spire (Fig. 1) results from three fac­ lels of adjacent layers are oriented generally perpendicularly tors. ach other, further strengthening the shell in all planes (18, (t) Shell dissolution does not occur in about the jKisterior 25% of the aperture length (indicated by the dark pigment bands 'he outer lip or growing edge of the shell consists only of in layer 2 of the spire; Fig. 1, i); iostracum and outermost calcified layer (layer 1); the long (a) A broad adhesion zone between successive whorls (Fig. .5 of the primary lamels of layer 1 lie generally in the plajie 1) formed by deposition of layer 1 on the outside of the ig- 2E. Layer 2 begins within 10° of the outer lip; the long underlying layer 1 at the posterior end of the aperture; depo­ ; of its primary lamels parallel the outer lip (Figs. 2 A and sition of layers 2 and 3 fills the space just anterior to the recurved ‘ Laver 3 begins about 30-50° from the outer lip; its primary layer 1 (Fig. 1, ii); this filling is complete about 40° from the , els are oriented as in layer 1 (Fig. 2E). Layer 4 (Figs. 3 and outer lip; be^ ^ns about 90° from the outer lip, but occurs only in the (in) Deposition of layer 4, starting at about 260°, on the terioi third of the shell; its primary lamels are generally abapical surface of shell that has remained undissolved in the •nted vXs in layer 2, but the crystal architecture of its inner previous whorl (Fig. 1, Hi). tion is often less clearly organized (Fig. 4D). At 180° intervals on projections of axial sections such as Fig. 1-1__« / r ______-I. ______I l y ______t ' fl. 4. C. twidus. Cross sections of shells showing crystal architecture and dissolution of internal walls at about 80% level (Fig. 1). (A) Between and 400°. Primary lamels in layers 1 and 3 are approximately parallel and those in layers 2 and 4 are approximately perpendicular to the of the section. Dissolution is proceeding from left (inward, toward cxjlumella) to right (outward, toward outer lip) along the exterior (abaxial) ice (upper right). Arrowhead indicates point of disappearance of layer 1 and beginning of thinning of layer 2. (B) Between 475° and 495°. iwhead indicates point of disappearance of layer 2 and beginning of thinning of layer 3. Spiral lines through layers 3 and 4 probably indicate )orary growth cessations. (A and B, light micrographs of etched, polished, gold-coated sections; scale bars = 0.5 mm. (C) At 475°. Specimen ted to show Ihe dissolution surface (dis) smoothly crossing axes of crystals. Arrowhead indicates point of disappearance of layer 2. (D) At *. Detail of layers 3 and 4, showing curvature of primary lamels in layer 3. (C and D, scanning electron micrographs of fractured, gold-coated imens; scale bars = 100 ^m.)

a estimated the area of shell added to the spire, the total area Hi) contributed the remainder. Of all shell material secreted lell secreted, and the area later dissolved. We then calcu- during these animals’ lives, 24% and 26%' had been dissolved ,1 ratios of these values raised to the 3/2 power to approxi- at the time of death. Inward of the point where shell thinning ? shell volume or mass. For two specimens of C. lividus 36 begins, this dissolution provided 70% and 60% of the living ,42 mm long, the intact region posterior to the dissolution space within the two shells. An alternative method of calcula­ (factors i and ii) accounted for 71% and 73%, respectively, tion, based on treating the projections of spire and last whorl

, .j. in preceding page). Thickness of the last and penultimate whorls and their component layers in C. lividus, to illustrate regions |ell '.th and dissolution. (A-D) Total shell thickness of three specimens from Hawaii (each indicated by a different symbol and line), ,ured hom cr vss sections at 32, 50,64, and 80% of aperture height (Fig. 1). (E-H) Proportion of total shell thickness represented by each \id^' * ^ corresponding to A-D. The lines plot mean values of the three specimens; numbers on the lines indicate layers. X intercepts at icatt initiation of each layer; X intercepts at right indicate disappearance of la.yers due to dissolution. 3410 Evolution: Kohn a/. Proc. Natl. Acad. Sci. USA 76 (1979)

DISCUSSION Gre^oire, C. (1972) in Chcmical Zoo/ogy, eds. Florkin, M. j Scheer. B. T. (Academic, New York, NY), Vol. 7. pp. 45-102 An evolutionary trend toward increasing thickness of marine Witbiir, K. M (1972) in Chemical Zaologij. eds. Florkin, M .. gastropod shells in the late Mesozoic and early Tertiary was Seheer, B. T. (Academic. New York, NV). \ ’ol. 7, pp. IIW- 14' associated with increasing intensity of predation by shell Clark, G. R. (1976) Am. Zool. 16, 617-626. ^ crushing (20). Conus, which first appears in the fossil record Watabe, N. & Wilhur, K. M., ctls. (1976) The Mcckanisms ’ in late Cretaceous (N. F. Sohl, personal communication), Mineralization in the Inoertehrntes and Plants (Univ. of Sou spearheaded this trend. Its thick last whorl and spire clearly Carolina Press, Columbia, SC). Cliarriker. M. R. (1972) \'e/igrr 15> 69-74. enhance protection from predation (18). However, the walls Morton, J. E (195,5) Proc. Zoo(. Soc. London 125. 127-168. • of the penultimate and earlier whorls are no longer exposed Vermeij. G. J. (1973) Mar. Biol. 20, 319-346. (Fig. 2B), and shell material can thus be dissolved from these Schroter, J S. (1783) Vehcr den inticrn Bau Her See- uml einif. inner whorls without reducing protection. auslandiscben Erd- und Flussschnecken (Varrentrapp Sohn We conclude that the internal shell remodeling we have Wenner, Frankfurt. Germany). described is adaptively significant to Conus in two ways: Cossmann, M (189(^) Essai? de paleoconchologie compart^e (^ Cossmann, Paris'!, Vol. 2. , (t) Shell weight is reduced while thickness of the last whorl 10. Kohn, A, J. & Rigps. .\. C. ()975)Si/sf. Zool. 24,346-359. and spire is maintained or enhanced in defense against attack 11. Timmermans, L. P. M. (1969) Nelh. J. Zool. 19,417-523. by enemies and physical stresses of the reef environment, 12. Weiner, S. &■ Hood. L, (1975) Science 190,987 989. and 13. MacClintock, C. (1967) Bull. Pealxidy Mm. Nal. Hist. Yo/p I n (it) More space is provided for the animal’s body within the 22,1-140. ' shell. This is especially important in Conus because of the 14. Bfiggild, O. B, (1930) Kgl. Danske Videnskab. Selskahs, S typical feeding mode of swallowing large, intact pre> organisms NatuTvidenskah. Math. A fdel 2,232-325. 15. Carter, ]. G. (1976) Dissertation (Yale, New Haven, CT), (21) and of the constraints of a conispiral shell that has a long, 16. Ciirrey, J. D. & Taylor. ]. D. (1974) J. Zool. 173, 395-406. narrow aperture and grows isometrically. 17. Philippon, J. & Plaziat, J, C. (1975) C.fi. HeM. Sci. Ar/sd. Sci. 2; 617-620. ' We thank Alan C. R i^s for technical assistance and discussion of 18. Currey. J. D. & Kohn, .A. J. (1976) J. Mat. Sci. 11,16)5-162 the manuscript and Arnold G. Schmidt for aid with the scanning 19, Philippon, J. (1974) C. B. Hehd. Sci. Acad. Sci. 279, 145-14' electron microscopy. This research was supported b\' National Science 20. Vermeij. G. J. (1977) PaleobioLogij 3, 245-258. Foundation Grants GB-32105X and DEB 77-24430. 21 . Kohn. ]. (1959) Ecol. Monogr. 29,47-90,