The Calcareous Egg Capsule of the Patagonian Neogastropod Odontocymbiola Magellanica: Morphology, Secretion and Mineralogy

THE CALCAREOUS EGG CAPSULE OF THE PATAGONIAN NEOGASTROPOD ODONTOCYMBIOLA MAGELLANICA: MORPHOLOGY, SECRETION AND MINERALOGY

GREGORIO BIGATTI1, MAXIMILIANO GIRAUD-BILLOUD2, ISRAEL A. VEGA2, PABLO E. PENCHASZADEH3 AND ALFREDO CASTRO-VAZQUEZ1,2 1LARBIM, Centro Nacional Patagońico (CENPAT-CONICET). Bvd. Brown 2915, U9120ACV Puerto Madryn, Chubut, Argentina; 2Laboratorio de Fisiologıá (IHEM-CONICET), Departamento de Morfologıá y Fisiologıá (FCM-UNCuyo), Casilla de Correo 33, 5500 Mendoza, Argentina; and 3Museo Argentino de Ciencias Naturales Bernardino Rivadavia (MACN-CONICET). Av. Ańgel Gallardo 470, Buenos Aires, Argentina

Correspondence: A. Castro-Vazquez; e-mail: [email protected] (Received 12 June 2009; accepted 1 March 2010) Downloaded from ABSTRACT Odontocymbiola magellanica is the only known South American volutid gastropod that deposits calcareous egg capsules. The spawn is moulded and ﬁxed to ﬂat or convex surfaces by the female’s ventral pedal gland, during an hours-long process in which the female adopts a stereotyped posture and appears nonreactive to most external stimuli. Microscopically, the different cells of the ventral http://mollus.oxfordjournals.org/ pedal gland show features suggesting their participation in the secretion of both the organic matrix and the calcium component of the calcareous layer. The latter consists mainly of numerous spher- spherulites that are packed together around cylindrical, septated spaces which traverse the spher- spherulitic layer and attach to the membranous layers surrounding the capsule cavity. These septated spaces should ensure permeability of the capsule wall, which is necessary for gas exchange and excretion by the embryo. The calcareous layer is made of high-magnesium calcite, a calcium carbonate polymorph in which Ca is partially substituted by Mg in the calcite lattice. Mg substitution is thought to confer a greater crack resistance to the mineral; it is found in many invertebrates, but

apparently has not been reported before in molluscs. Odontocymbiola magellanica is a long-lived species, at Indiana University Bloomington Libraries on May 12, 2015 investing heavily in its egg capsules.

INTRODUCTION Magallanes Strait and the Beagle Channel, reaching Chiloe´ Island on the Pacific coast of Chile (428S) (Weaver & Dupont, Most neogastropods retain their eggs within some form of 1970; de Castellanos & Landoni, 1992; Gallardo & closed capsule (Ponder, 1973; d’Asaro, 1986; Pechenik, 1986; Penchaszadeh, 2001; G.B., personal observation). Smith, Black & Shepherd, 1989; Knudsen, 1994, 2000; The current paper is part of an integrative investigation on Miloslavich, 1996a, b; Przeslawski, 2004; Pastorino, the biology of this snail, and aims to describe egg-laying be- Penchaszadeh & Scarabino, 2007). With the notable exception haviour, predation on the egg capsules, the anatomy and of the free and demersal egg capsule of the volutid Adelomelon calcium concentration of the ventral pedal gland which brasiliana (Penchaszadeh et al., 1999), the capsules are fixed to moulds and secretes the calcified layer, and the structure and some substrate and moulded by a ventral pedal gland. They mineral composition of the capsule itself. occur in a wide variety of forms, both in the intertidal and the subtidal zones. Although their primary function is thought to be protection of the developing embryo, these capsules should MATERIAL AND METHODS be organized in a way that does not impede embryonic nutri- Field work tion, gas exchange and excretion, and finally they should permit the hatchlings to exit (Rawlings, 1999). Field observations and sampling were conducted monthly, South American Volutidae include 12 genera with about 30 from June 2001 to September 2005 by SCUBA diving, at species, but only some of their egg capsules have being investi- depths between 5 and 20 m during low tides, in Golfo Nuevo, gated (De Mahieu, Penchaszadeh & Casal, 1974; Bandel, Argentine Patagonia (428430S; 658010W). Egg capsule-laying 1976; Penchaszadeh & De Mahieu, 1976; Penchaszadeh et al., and -moulding behaviour of Odontocymbiola magellanica was 1999; Luzzatto 2006; Clench & Turner, 1970; Penchaszadeh & observed underwater and photographs were taken. Egg cap- Miloslavich, 2001; Miloslavich et al., 2003; Bigatti et al., 2009). sules at different stages of development were collected in the Odontocymbiola magellanica (Gmelin, 1791) is the only South same area, sometimes directly from laying females. The colour, American volutid known to deposit calcareous egg capsules transparency and toughness of each collected egg capsule were (Penchaszadeh et al., 1999), but the morphology and mineral- recorded. Also, predators of egg capsules and the predated cap- ogy of the capsules, and egg-laying behaviour, have not been sules were collected and identified. studied. Odontocymbiola magellanica, the Patagonian red snail, is found Anatomy of the pedal gland from the shallow subtidal to depths of more than 800 m (Penchaszadeh & De Mahieu, 1976) and ranges through Ventral pedal glands of mature egg-laying and nonegg-laying the Magellanic biogeographic province, from 358S on the females were observed and photographed in the field. Slices of Argentine Atlantic coast (off Rio de la Plata) down to the pedal gland tissue and tissue from the periglandular sole of

Journal of Molluscan Studies (2010) 76: 279–288. Advance Access Publication: 15 April 2010 doi:10.1093/mollus/eyq006 # The Author 2010. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. G. BIGATTI ET AL. capsule-moulding females were fixed in either Bouin’s fluid or RESULTS 2.5% glutaraldehyde in filtered sea water, embedded in resin (either Leica Historesinw or Spurr resin), sectioned and stained Egg capsule laying and predation in the field with either haematoxylin and eosin or toluidine blue. In some Female Odontocymbiola magellanica choose flat or convex surfaces other cases, they were fixed in 5% formaldehyde in seawater, for attachment of egg capsules. The most frequently used embedded in paraffin and stained with von Kossa’s silver objects were stones, bivalve shells and elasmobranch egg cap- nitrate procedure for histochemical Ca detection (Howard sules, but they may also attach the capsules to the substrate et al., 2004). beneath the sand layer. At the height of the season (December) egg-laying females may be seen grouped in female-only aggregates of 8–15 individuals. The female grasps Description, and transmission and scanning electron microscopy the chosen object with its foot (Fig. 1A), and it spawns a of egg capsules white, soft and pliable capsule (Fig. 1C, inset). It then encloses the capsule beneath its foot and adopts a stereotyped posture Capsular height and width were measured with callipers for 52 (Fig. 1B). The spawn is moulded and fixed to the substrate by capsules, and the volume of their internal content was the ventral pedal gland during this period, after which the measured in a graduated cylinder to the nearest 1 ml. female looses its hold and leaves behind a white, hard, round Downloaded from Lateral fragments (3 mm wide) of recently laid egg capsules bell-shaped capsule attached to the substrate object (Fig. 1C). were cut and fixed for 3 h in 2.5% glutaraldehyde in 1 M When taken to the laboratory, these capsules react to hydro- phosphate buffer (pH 7.4), postfixed overnight with 1% chloric acid, producing gas bubbles, which suggests carbonate osmium tetroxide in the same buffer, stained with 2% uranyl in the external cover. acetate for 45 min, dehydrated via graded ethanol and The capsule-laying posture of O. magellanica is characterized acetone, and finally embedded in Spurr’s resin and sectioned by a tensing of the foot which becomes approximately conical, http://mollus.oxfordjournals.org/ in a ultramicrotome for transmission electron microscopy tilting the long axis of the shell about 458 from the horizontal (TEM). For topographic orientation, 1-mm sections were plane (Fig. 1B). The siphon is only partly retracted, while the stained with 1% toluidine blue in 1% potassium carbonate for head and nuchal lobes are relaxed and flattened, exposing the 30 s. Later, silver grey sections were observed with a Philips eyes. However, the laying female shows an extremely low reac- EM 301 TEM. Also, similar 5 mm wide samples of the same tivity to the diver’s proximity or to any disturbances. If capsules were fixed as described above, but they were critical- grasped by the diver and taken out of water, it remains point dried, metal coated and observed under a Philips XL 30 attached to the spawning substrate, and if forcefully detached scanning electron microscope (SEM). from the substrate object, it will not retract into the shell and maintains the capsule-moulding depression in its foot for several minutes (Fig. 2A). Though we could not establish at Indiana University Bloomington Libraries on May 12, 2015 Calcium determination in haemolymph, ventral pedal gland exactly the duration of egg capsule laying episodes in O. magel- and extra-glandular mucosa lanica, our field observations suggest that it may take several hours. After cracking the shell, haemolymph was obtained from the During these field observations, predation on capsules by the heart of capsule-moulding females (haemolymph from two to green sea urchin Arbacia dufresnii and the starfish Cosmasterias three females was pooled to obtain samples of at least 1 ml). lurida was observed. Predation occurs on capsules both at the Samples of about 300 mg of the ventral pedal gland and the beginning of development, when the calcareous cover is intact, extra-glandular mucosa were dissected with small scissors. and near the end of development, when this cover has often Samples were homogenized with 0.1 N hydrochloric acid, cen- deteriorated (see below). trifuged at 13,000 G for 5 min, and the supernatants were kept at 2808C until the day of determinations. Total calcium was measured in 500-ml aliquots of the supernatants, which were Anatomy of the ventral pedal gland exposed to o-cresolftalein in alkaline solution (Schwarzenbach, 1955); the resulting complexone was measured spectrophoto- The gland located in the mid-anterior part of the sole of the metrically; the sensitivity of the method was 0.2 mg per tube. foot appears as a well-delineated, dark and grooved mucosa, which allows recognition of females in the field, since it is absent in males (Fig. 2B). As noted above, when egg capsule Mineralogy of capsule wall deposition is forcefully interrupted, the gland appears as a deep depression that corresponds exactly to the shape of the The calcareous wall of white, recently laid egg capsules was capsule which was being moulded (Fig. 2A). scraped, and the obtained material was air dried and pow- Microscopically, the ventral pedal gland is composed of dered in a mortar, then analysed with a Siemens D 5000 dif- three distinct layers: superficial, intermediate and deep fractometer using Cu/Ni radiation. The diffractograms were (Fig. 3A). The intermediate layer is an almost continuous compared with those of calcite and magnesium calcite space of haemocoelic cavities, muscle fibres and some connec- standards. tive tissue bands which separate the external epithelium Egg capsule powder was resuspended in 2.5% sodium hypo- (superficial layer) from apparently ‘subepithelial’ cell aggre- chlorite, washed and dried under vacuum for 7 days at room gates (deep layer). temperature. The sample was then treated with 50% hydro- The superficial layer is mostly composed of ciliated and chloric acid, dried, dissolved in 2% hydrochloric acid, paper goblet cells. Most ciliated cells have a slender nucleus with filtered, diluted with an equal amount of distilled water and finely granular chromatin and no apparent nucleolus. Goblet finally measured in triplicate with a Unicam atomic absorption cell nuclei may either be dark and located near the basal mem- spectrometer. Also, elemental analysis of hypochlorite-treated brane, or similar to those of ciliated cells. In general, the calcareous material obtained from a recently laid capsule was secretory content of goblet cells is faintly basophilic when the made by SEM (Jeol JSM 6460 LV) equipped with an EDAX secretory vacuole is basally located, but apical vacuoles are PW 7757/78 X-ray energy scattering microanalyser, for stan- usually more intensely basophilic (Fig. 3B). On the external dardless element quantification. surface, the apices of goblet cells often occupy empty spaces in

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Figure 1. A. Female Odontocymbiola magellanica grasping a bivalve shell from its convex side for egg capsule deposition and moulding. B. A capsule-moulding female, assuming the typical stereotyped posture; note that shell is tilted some 458 from the ground, siphon is partially retracted (white arrow), and left tentacle and nuchal lobes can be observed (black arrows). C. A recently moulded capsule that was fixed to a bivalve shell (Aequipecten tehuelchus); the inset shows a soft and unmoulded capsule that was released by a female after being taken out of the water. D. An egg capsule at the end of development; the calcareous covering has been lost, and the membranous capsule allows the prehatching juveniles to be seen within. the mass of cilia. Calcium detection by von Kossa’s silver- ciliated and goblet cells (Fig. 3D). Von Kossa staining also staining method showed a finely granular, dark brown deposit showed calcium in the goblet cells of the periglandular epi- concentrated in the secretory vacuoles of the goblet cells thelium (Fig. 3F). The underlying connective tissue is traversed (Fig. 3E). An intriguing aspect of the superficial layer is the by muscle fibres that are perpendicular to the surface epithelium. occurrence of isolated groups of rather large oval nuclei showing heavy heterochromatic clumps; most but not all of these nuclei Calcium concentration in the ventral pedal gland are located at the base of the superficial layer (Fig. 3C). The deep layer is formed by bundles of long, bottle-shaped and the extra-glandular mucosa cells whose necks cross the intermediate layer (Figs 3A–D) and Mean calcium levels (+SE, n ¼ 6) in both the glandular and become intermingled with cells of the superficial layer, but extra-glandular sole tissue were similar (1.21 + 0.08 and whose cell bodies form elongated aggregates, sometimes 0.97 + 0.15 mg/ml, respectively) while the mean value (+SE, around pseudofollicular cavities. These aggregates are perpen- n ¼ 6) in the haemolymph was much lower (0.070 + 0.007 mg/ dicular to the epithelial surface and their cells have a darkly ml), suggesting a similar ability of both pedal tissues to take up basophilic, granular cytoplasm (Fig. 3A, D). Occasionally, a and concentrate calcium from the haemocoel. bunch of the long necks of the bottle-shaped cells can also be recognized in the superficial layer. The ventral pedal gland is sharply delimited from the sur- Shape and size of egg capsules rounding epithelium of the foot by a narrow groove (Fig. 3D). Although there was a common rounded bell shape, the cap- The periglandular epithelium of the sole has a single layer of sules showed a large variation in height (range, 18–41 mm;

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Figure 2. A. A female Odontocymbiola magellanica forceably detached from a stone on which it was moulding an egg capsule; note the depression in the sole of the foot (arrow), which is lined by the ventral pedal gland epithelium, and which will be maintained for several minutes. Another egg capsule, previously deposited on the same stone, is seen to the right. B. The sole of a mature female which has been moulding an egg capsule, but whose foot is already relaxed, shows the shallow but well-defined depression of the ventral pedal gland (arrow); the egg capsule laid by this female is also shown. at Indiana University Bloomington Libraries on May 12, 2015 mean + SD, 29.2 + 6.4 mm) and width (range, 20–39 mm; useful preparations of the inner aspect of the membrane, mainly mean + SD, 30.0 + 5.2 mm). The volume of the internal con- because of the abundant intracapsular fluid materials that tents was also variable (range, 3.6–29.7 ml; mean + SD, remained attached to it, so that we could not establish if the 13.2 + 7.3 ml). Height and width of egg capsules were not cor- apparent pores were indeed traversing the membrane. related (r2 ¼ 0.408; P ¼ 0.639). By the end of intracapsular development, the outer calcareous layer usually becomes weakened, mainly as a consequence of digestion of the organic septated spaces, which appears Structure and mineral composition of the egg capsule associated with the development of an epibiotic community Figure 4 is a schematic drawing of the organization of the within (Fig. 5E, F). SEM observations show patches of a het- capsule layers in recently deposited capsules. SEM showed that erotrophic bacterial mat, diatoms and probably filamentous the capsule wall is formed by a calcareous cover (which may cyanobacteria and/or fungi (Fig. 5E, F). Eventually, the cal- split into an inner and outer layer of different structure, see careous layer may disappear, leaving only the epibiotic com- below) and by at least two inner membranous layers (Fig. 5A). munity on the translucent membranous wall, through which The calcareous layer is covered externally by a thin organic crawling embryos may be seen within (Fig. 1D). cuticle and consists of numerous spherulites (10–20 mm width; TEM showed that the membranous layers are composed of Fig. 5B) packed together in an organic matrix around sep- an amorphous organic matrix of low and rather uniform elec- tated, approximately cylindrical spaces (Fig. 5B, C). These tron density and of numerous protein fibrils of higher electron spaces traverse the entire calcareous layer at more or less density. The latter are more densely arranged in the outer regular intervals (Fig. 5C) and their septa are mostly horizon- than in the inner layers (Fig. 6A, B). tal, though some vertical septa also occur (Fig. 5B). The sep- The X-ray powder diffraction pattern obtained from tated spaces and their spherulitic walls lie on a thin layer of recently laid egg capsules showed that the calcareous material microfibrillar crystallites (Fig. 5A), which are in turn attached was magnesium calcite, a calcium carbonate polymorph to the outer membranous layer, and represent the first calcar- (Fig. 7A). Atomic absorption analysis indicated that 21.1% of eous material deposited by the female. the Ca was substituted by Mg in the calcite lattice. EDAX When detached from the microfibrillar calcareous layer, standardless estimations in a similar sample (Fig. 7B) yielded a the underlying membranous layer has a patchy appearance somewhat smaller figure of Mg substitution (13.4%). (30–90 mm wide patches; Fig. 5D). Some imprints on these patches suggest that some of the septated spaces may traverse the microfibrillar calcareous layer and rest directly on the mem- DISCUSSION branous outer layer (Fig. 5D). Numerous apparent pores, about Egg-laying behaviour 1 mm in diameter, each surrounded by an elevated ridge, are irregularly distributed over the membrane surface (Fig. 5D). The female Odontocymbiola magellanica shows low reactivity Occasionally, remnants of the inner calcareous layer are seen during the egg-laying process, which would appear to expose it still attached to the membrane (Fig. 5D). We could not obtain undefended to predators. This risky parental investment may

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Figure 3. A. Microscopic organization of the ventral pedal gland in female Odontocymbiola magellanica. Three layers (superficial, intermediate and deep) can be recognized. The necks (black triangles) of the bottle-shaped cells cross the intermediate layer and intermingle with cells in the superficial layer; asterisks indicate elongated aggregates of bottle-shaped cells, which are delimited by acidophilic muscular bundles; haemocoelic spaces appear as clear spaces in the intermediate layer, where the dark nuclei mostly belong to haemocytes. B. Superficial layer and part of intermediate layer of the ventral pedal gland; black arrows indicate the basophilic content of upper vacuoles of goblet cells; empty spaces between the ciliary tufts probably indicate the openings of goblet cells to the surface; clear haemocoelic spaces and haemocytes are seen in the intermediate layer. C. Another view of the superficial layer and part of the intermediate layer showing a group of large oval nuclei (demarcated by white triangles). D. The narrow groove which separates the ventral pedal gland (upper part of the micrograph) from the surrounding epithelium of the sole (lower part), which also shows ciliated and goblet cells; the underlying lamina propria shows muscular bundles that are perpendicular to the surface. E. Von Kossa staining of goblet cells in the surface epithelium of the ventral pedal gland. A finely granular silver deposit indicates calcium in the vacuoles of the apical parts of goblet cells (black arrows), while a less conspicuous deposit also appears in basal vacuoles (white arrows). F. Von Kossa staining of goblet cells in the epithelium surrounding the ventral pedal gland. A similar distribution of silver deposits indicates that calcium is also secreted by this epithelium. Arrows as in E. Abbreviations: c, cilia; d, upper part of the deep layer; g, goblet cells; i, intermediate layer; n, nuclei of ciliated cells; s, superficial layer. Scale bars: A ¼ 100 mm; B ¼ 50 mm; C ¼ 20 mm; D ¼ 100 mm; E ¼ 20 mm; F ¼ 20 mm.

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layers are not separate entities (as an ‘epithelium’ and a ‘sube- pithelium’), but a single epithelium with cell bodies at different levels. Similar structures have also been found in the copulatory apparatus of the architaenioglossan caenogastropod Pomacea canaliculata (Gamarra-Luques et al., 2006). In addition, a less extreme case of submerged epithelium has been studied in detail in the albumen gland duct of P. canaliculata, where TEM has shown that the folds of the basal lamina follow each submerged cell (or cell group), conﬁrming that the superﬁcial and deep layers constitute a single epithelium (Catala´ n, Ferna´ ndez & Winik, 2002). Bottle-shaped cells in the deep layer are loaded with a basophilic granular cytoplasm, which is likely to participate in the secretion of the organic matrix of the calcareous layer. Calcium secretion appears associated not with bottle-shaped

cells in the deep layer, but with surface goblet cells, both in Downloaded from the pedal gland itself and in the surrounding foot epithelium, which may contribute to generating the supersaturated calcium solution needed for deposition of the calcium covering.

Egg capsule shape and function http://mollus.oxfordjournals.org/ Neogastropods have evolved a wide variety of egg capsules, whose shape has systematic significance (Ponder, 1973) and Diagram of the structure of the wall of a recently moulded Figure 4. whose primary function is thought to be protection of the egg capsule of Odontocymbiola magellanica (see also Fig. 5). Organic structures are shown in white and indicated with letters. Most developing embryo from predators and physical stresses calcareous structures (shown in grey) are magnesium calcite spherulites (Rawlings, 1999). with an inner radiated acicular structure (this is not represented in the In the case of O. magellanica, the round, bell-shaped egg cap- figure); a basal and mostly continuous layer is made of an amorphous/ sules vary greatly in both absolute size and proportions (no cor- acicular calcareous material, which is attached to the outer relation was found between height and width in the present membranous layer. Abbreviations: b, basal calcareous layer; c, external study), so that these attributes may have little systematic value. cuticle; i, inner membranous layer; o, outer membranous layer; s, The subtidal benthic deposition of these capsules protects them membranous septated spaces. from physical stresses, which certainly affect the spawn of interti- at Indiana University Bloomington Libraries on May 12, 2015 dal species (stresses include desiccation, osmotic stress, tempera- have the functional significance of creating a sealed space ture changes and ultraviolet radiation; Rawlings, 1999). Their between the female’s foot and the laying substrate. This space tough calcareous covering should make them more resistant to may serve a function similar to the ‘extrapallial’ space during predators, particularly since O. magellanica shows a protracted shell formation (Wilbur & Saleuddin, 1983), i.e. a space in (and direct) intracapsular development (about 2 months, which the secreted organic matrix and a supersaturated Bigatti, 2005). Notably, Perron (1981) has shown for 10 calcium solution provide the conditions for formation of Hawaiian Conus species, that capsule toughness directly correlates calcium carbonate crystallites. The efficiency of this capsule- with developmental length. Another feature that may correlate secreting process is remarkable since it occurs rapidly, in hours, with the long residence time of embryos within the capsule in O. while shell secretion is a process taking days or weeks. magellanica is that the intracapsular fluid contains more nutrients Female O. magellanica attach their egg capsules to a variety of than in other South American volutids (Bigatti, 2005). submerged objects, but they always choose flat or convex surfaces for attaching them. It is intriguing that females of The calcareous cover Adelomelon ancilla, which is sympatric with O. magellanica in the study area, always choose concave structures for capsule depo- Considering the potential protective value, it is intriguing that sition. Penchaszadeh et al. (1999) recorded an illustrative heavily calcified egg capsules such as those of O. magellanica example of this divergent behaviour, in which two capsules and Alcithoe arabica (Ponder, 1970) appear only rarely among were laid on the same bivalve shell: the one of O. magellanica on the Neogastropoda. The wall of some neritimorph egg capsules the convex surface, and that of A. ancilla on the concave side. may incorporate calcareous particles of varying origins (e.g. Andrews, 1936; Berry, 1965; Smith et al., 1989; Tan & Lee, 2009), but in general heavily calcified capsules are mainly Anatomy of the pedal gland found in the Stylommatophora (Tompa, 2005) and freshwater Architaenioglossa that deposit aerial eggs (Hayes et al., 2009). The distinctive shapes of neogastropod egg capsules are the Therefore, calcareous egg capsules appear mainly as cases of result of a moulding process that occurs within a cavity formed convergent evolution associated with the colonization of terres- by the ventral pedal gland, located in the sole of the female’s trial and freshwater habitats. foot (Rawlings, 1999). Histologically, the gland which moulds A surprising discovery is what we believe is the first record of the capsules in O. magellanica, and which deposits the calcar- high-magnesium calcite in molluscs. This mineral is a calcium eous layer, consists of a ‘submerged’ epithelium similar to those carbonate polymorph in which Ca2þ is randomly substituted found in other invertebrates (Welsch & Storch, 1976), showing to some extent by Mg2þ in the calcite lattice; Mg substitutions a surface layer composed of ciliated and goblet cells and a over 4% (values found in O. magellanica capsules were 13.4– deep, apparently subepithelial layer. The deep layer in O. 21.1%) are considered to be high (Ries & Blaustein, 2003). magellanica is composed of large, elongated, bottle-shaped cells, Gastropod shells are made of aragonite and to a lesser extent which makes it an extreme case of a submerged epithelium. As calcite plus aragonite, whose crystallites are embedded in an noted by Welsch & Storch (1976), the superficial and deep organic matrix (Watabe, 1988; Bandel, 1990). Many varied

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Figure 5. SEM of the egg capsule wall of Odontocymbiola magellanica (see Fig. 4 for general orientation). A. Basal calcareous layer together with the outer and inner membranous layers have been detached from the spherulitic calcareous layer (visible in the background). Numerous holes corresponding to the membranous septated spaces are seen at the base of the spherulitic layer. B. Vertical cut trough the calcareous spherulitic layer shows several horizontal septa which separate chambers in the vertically oriented cylindrical spaces (white arrows); asterisks indicate the walls formed by piled-up spherulites, which separate the septated spaces. C. External aspect of the egg capsule wall in which cuticle has been torn out during critical-point drying, exposing numerous calcareous spherulites, and the upper chambers of two septated spaces (black arrows); in the upper one a horizontal septum forms the chamber’s floor, while five vertical septa divide the upper chamber of the lower septated space. D. Irregular pavement appearance of the external aspect of the outer membranous wall, showing the likely imprints of the units formed by a septated space and its surrounding spherulitic wall; portions of the basal calcareous layer have remained attached to the membrane; bubble-like imprints probably correspond to the base of septated spaces which have traversed the basal calcareous layer (black arrows). The white arrows indicate apparent pores surrounded by an elevated ridge. E. Deteriorated calcareous wall showing diatoms (white arrows) within the spaces left by degradation of septated cavities. The asterisk shows the membranous remains of the wall of a formerly septated space. F. Deteriorated calcareous wall showing several diatoms (white triangles) and a filamentous budding organism (left white arrow); the right white arrow points to a spherulite in which dissolution of the organic surface reveals the inner acicular structure. Abbreviations: bc, basal calcareous layer; c, cuticule; h, holes; im, inner membranous layer; om, outer membranous layer. Scale bars: A ¼ 200 mm; B, C ¼ 50 mm; D, E ¼ 20 mm; F ¼ 20 mm.

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Figure 6. TEM sections of the outer (A) and inner (B) membranous layers of the egg capsule wall of Odontocymbiola magellanica. Protein ﬁbrils

embedded in a matrix of low electron density appear more densely packed in the outer than in the inner membranous wall. Scale bars ¼ 1.5 mm. Downloaded from and intricate crystallite structures are formed (see Watabe, that a ‘sclerotization’ of the membranous wall is caused by 1988, and Bandel, 1990, for references) which confer toughness pedal gland secretions. and resistance to the intrinsically brittle nature of calcium

carbonate. http://mollus.oxfordjournals.org/ Calcified gastropod egg capsules that have been studied are also composed of either aragonite or calcite (Bandel, 1990). An Seasonality of egg-laying behaviour exception is the aerial egg capsules of the architaenioglossan The egg-laying behaviour of O. magellanica is only observed genus Pomacea, where vaterite is found (Hall & Taylor, 1971; during the Southern winter and spring. In general, it has been Meenakshi, Blackwelder & Watabe, 1974; Catala´ n et al., thought that water temperature and food availability are of 2008). Spherulites in the calcareous capsules of O. magellanica greater importance than day length as cues for the onset and (Figs 4, 5B, C, F) are somewhat similar to those of vaterite termination of reproductive processes in aquatic animals (Daly under the SEM, but powder X-ray diffraction analysis of the & Wilson, 1983). This view has been confirmed by obser- material showed a pattern like that of a magnesium calcite vations in many caenogastropod species (e.g. Giese & Pearse, standard (Fig. 7). 1974; Fretter & Graham, 1994) and it has been experimentally Magnesium calcite occurs in either shells or skeletal struc- (and quantitatively) demonstrated in an architaenioglossan at Indiana University Bloomington Libraries on May 12, 2015 tures of a variety of nonmolluscan groups, including sponges caenogastropod (Albrecht, Carrenõ & Castro-Vazquez, 1999; (Jones & Jenkins, 1970), sea urchins (Magdans & Gies, 2004), Albrecht et al., 2004). The reproductive cycles of volutid snails seastars (Gayathri et al., 2007), serpulid worms (Neff, 1969), living on the Atlantic coast of Argentina have been reported crustaceans (Neues et al., 2007) and ascidians (Aizenberg et al., for two species that live further north (358S) than O. magella- 2002). It is thought that magnesium substitution may impart a nica: Zidona dufresnei (Gime´ nez & Penchaszadeh, 2002) and greater crack resistance to the calcite (Magdans & Gies, 2004). Adelomelon brasiliana (Cledo´ n, Arntz & Penchaszadeh, 2005) Permeability of the egg capsule layer (which is required for and the observed seasonal reproduction also support this gas exchange and excretion by embryos) is assured by diffusion general view. Notwithstanding, Bigatti, Marzinelli & through the membranous septated spaces and the membranous Penchaszadeh (2008) have reported that the onset of capsule- inner capsule layer, and probably by pores in the latter laying behaviour of O. magellanica in Golfo Nuevo (428S) (Fig. 5D). Although we did not investigate the structure of the occurs in July, reaches its maximum frequency in December egg capsule when first spawned by the female, i.e., before and ceases in January (Southern summer). It seems, therefore, being moulded by the pedal gland (Fig. 1C, inset), it is likely that the annual rhythm of oviposition and capsule-moulding that some secretion or modification of the membrane occurs behaviour in this species is entrained to increase in day length, during the moulding process, as shown by Sullivan & Maugel and that this behaviour turns off in response to the small daily (1984) in the nassariid neogastropod Ilyanassa obsoleta. Also, decreases in day length that occur after the summer solstice, Price & Hunt (1973, 1974) have shown in Buccinum undatum even though water temperature continues to rise at that time.

Figure 7. A. X-ray diffractogram of the calcareous powder obtained from the egg capsule wall of Odontocymbiola magellanica. The nine peaks are as those of a magnesium calcite standard (indicated by vertical bars). B. EDAX analysis of the powder of the calcareous egg capsule, showing the count peaks for C, O, Mg and Ca.

286 EGG CAPSULES OF VOLUTID ODONTOCYMBIOLA

Conclusion magellanica (Gmelin, 1791) from Golfo Nuevo, Patagonia. Journal of the Marine Biological Association of the United Kingdom, 87: 755–759. Odontocymbiola magellanica is a remarkably long-lived species (up BIGATTI, G. & CIOCCO, N.F. 2008. Volutid snails as an alternative to 20 years old; Bigatti, Penchaszadeh & Cledo´ n, 2007) reach- resource for artisanal fisheries in Northern Patagonic gulfs. ing sexual maturity at 7–8 years old, and showing an extended Availability and first suggestions for diving catches. Journal of oviposition season ending sharply when days become shorter Shellfish Research, 27: 417–421. (Bigatti et al., 2008). Copulatory behaviour, however, is BIGATTI, G., MARZINELLI, E.M. & PENCHASZADEH, P.E. observed throughout the year and sperm are stored in a bursa 2008. Seasonal reproduction and sexual maturity of Odontocymbiola copulatrix (Bigatti et al., 2008). The slowly reacting female is magellanica (Neogastropoda: Volutidae) in Patagonian shallow exposed for hours to predators when laying an egg capsule con- waters, Argentina. Invertebrate Biology, 127: 314–326. taining 4–18 eggs and which it endows with both membranous BIGATTI, G. & PENCHASZADEH, P.E. 2005. Imposex in and calcareous coverings (the latter made of magnesium Odontocymbiola magellanica (Gmelin, 1791) (Mollusca: Gastropoda) calcite) and with a comparatively significant protein content in Patagonia. Comunicaciones de la Sociedad Malacolo´gica del Uruguay, 9: for embryo nutrition (Bigatti, 2005). Furthermore, the species 377–379. is prone to female masculinization (‘imposex’) and shows BIGATTI, G., PENCHASZADEH, P.E. & CLEDO´ N, M. 2007. Age, changes in shell morphology and female weight loss near har- growth and somatic production in the volutid gastropod Downloaded from bours, presumably as a consequence of environmental xeno- Odontocymbiola magellanica from Golfo Nuevo, Patagonia Argentina. biotics (Bigatti & Penchaszadeh, 2005; Bigatti & Carranza, Marine Biology, 150: 1199–1204. 2007, Bigatti et al., 2009). Therefore, O. magellanica appears as BIGATTI, G., PRIMOST, M.A, CLEDO´ N, M., AVERBUJ, A., a vulnerable, though commercially exploitable snail (Bigatti & THEOBALD, N., GERWINSKI, W., ARNTZ, W., Ciocco, 2008) whose interesting morphological, behavioural MORRICONI, E. & PENCHASZADEH, P.E. 2009. and ecophysiological features should assure the continued Contamination by TBT and imposex biomonitoring along 4700 km study of its biology. of Argentinean shoreline (SW Atlantic: from 368Sto548S). Marine http://mollus.oxfordjournals.org/ Pollution Bulletin, 58: 695–701. CATALA´ N, N.M.Y., FERNA´ NDEZ, S.N. & WINIK, B.C. 2002. ACKNOWLEDGEMENTS Oviductal structure and provision of egg envelopes in the apple snail Pomacea canaliculata (Gastropoda, Prosobranchia, Special thanks are due to Eugenia and Victoria Zavattieri and Ampullariidae). Biocell, 26: 91–100. to Oscar Wheeler for field assistance, and to Isabel Farı´ as CATALA´ N, M., BABOT, E., FERNA´ NDEZ, S. & WINIK, B.C. (IDNEU, Buenos Aires), Fabia´ n Tricarico (MACN, Buenos 2008. Ultrastructural and physicochemical characterization of Aires) and Jaime Groizard (Aluar S.A.I.C., Puerto Madryn) egg shells in Pomacea canaliculata. International Journal of Morphology, for help in electron microscope observations. EDAX elemental 26: 781. determinations were made through the courtesy of Aluar ´

CLEDON, M., ARNTZ, W. & PENCHASZADEH, P.E. 2005. at Indiana University Bloomington Libraries on May 12, 2015 S.A.I.C. (Puerto Madryn). This work was supported by grants Gonadal cycle in an Adelomelon brasiliana (Neogastropoda: from FONCYT (PICTR 01869 and PICT 2008-0323) and Volutidae) population of Buenos Aires province, Argentina. Marine CONICET (PIP 5301) to G.B., P.E.P. and A.C.-V., and from Biology, 147: 439–445. the National University of Cuyo (to A.C.-V.). CLENCH, W.J. & TURNER, R.D. 1970. The family Volutidae in the Western Atlantic. Johnsonia, 4: 369–271. D’ASARO, C.N. 1986. Laboratory spawning, egg membranes, and REFERENCES egg capsules of 14 marine prosobranchs from Florida and Bimini, AIZENBERG, J., LAMBERT, G., WEINER, S. & ADDADI, L. Bahamas. American Malacological Bulletin, 4: 185–199. 2002. Factors involved in the formation of amorphous and DALY, M. & WILSON, M. 1983. Sex, evolution and behaviour. Willard crystalline calcium carbonate: a study of an ascidian skeleton. Grant Press, Boston. Journal of the American Chemical Society, 124: 33–39. DE CASTELLANOS, Z.J.A. & LANDONI, N. 1992. Cata´logo ˜ ALBRECHT, E.A., CARRENO, N.B. & CASTRO-VAZQUEZ, A. descriptivo de la malacofauna marina magallańica. Neogastropoda, Volutidae, 1999. Quantitative study of environmental factors influencing the Volutomitridae, Cancellariidae, Olividae y Marginellidae. Informes de la seasonal onset of reproductive behaviour in the South American Comisio´ n de Investigaciones Cientı´ ficas de la Provincia de Buenos apple-snail Pomacea canaliculata (Prosobranchia: Ampullariidae). Aires, 10, La Plata. Journal of Molluscan Studies, 65: 241–250. ˜ DE MAHIEU, G.C., PENCHASZADEH, P.E. & CASAL, A. 1974. ALBRECHT, E.A., KOCH, E., CARRENO, N.B. & Algunos aspectos de las variaciones de proteı´ nas y aminoa´ cidos CASTRO-VAZQUEZ, A. 2004. Control of the seasonal arrest of libres totales del lı´ quido intracapsular en relacio´ n al desarrollo copulation and spawning in the apple-snail Pomacea canaliculata embrionario en Adelomelon brasiliana (Lamarck, 1811). Cahiers de (Prosobranchia: Ampullariidae). Veliger, 47: 147–152. Biologie Marine, 15: 215–227. ANDREWS, E.A. 1936. The egg capsules of certain Neritidae. Journal British prosobranch molluscs: their of Morphology, 57: 31–59. FRETTER, V. & GRAHAM, A. 1994. functional anatomy and ecology. Edn 2. Ray Society, London. BANDEL, K. 1976. Spawning, development and ecology of some higher Neogastropoda from the Caribbean Sea of Colombia. GALLARDO, C.S. & PENCHASZADEH, P.E. 2001. Hatching mode Veliger, 19: 176–193. and latitude in marine gastropods: revisiting Thorson’s paradigm in the Southern Hemisphere. Marine Biology, 138: 547–552. BANDEL, K. 1990. Shell structure of the Gastropoda excluding Archaeogastropoda. In: Skeletal biomineralization: patterns, processes and GAMARRA-LUQUES, C., WINIK, B.C., VEGA, I.A., evolutionary trends. Vol. I (J.G. Carter, ed.), pp. 117–134. Van ALBRECHT, E.A., CATALAN, N.M. & CASTRO-VAZQUEZ, Nostrand Reinhold, New York. A. 2006. An integrative view to structure, function, ontogeny and BERRY, A. 1965. Reproduction and breeding fluctuations in Hydrocena phylogenetical significance of the male genital system in Pomacea montemsutiana, a Malayan limestone archeogastropod. Proceedings of canaliculata (Caenogastropoda, Ampullariidae). Biocell, 30: the Zoological Society (London), 144: 219–227. 345–357. BIGATTI, G. 2005. Anatomıá, ecologıá y reproduccioń del caracol rojo GAYATHRI, S., LAKSHMINARAYANAN, R., WEAVER, J.C., Odontocymbiola magellanica (Gastropoda: Volutidae) en golfos MORSE, D.E., KINI, R.M. & VALIYAVEETTIL, S. 2007. In norpatagońicos. PhD thesis, Universidad de Buenos Aires, Argentina. vitro study of magnesium-calcite biomineralization in the skeletal BIGATTI, G. & CARRANZA, A. 2007. Phenotypic variability materials of the seastar Pisaster giganteus. Chemistry. A European associated with the occurrence of imposex in Odontocymbiola Journal, 13: 3262–3268.

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