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 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 spherspherulites that are packed together around cylindrical, septated spaces which traverse the spherspherulitic 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, investing heavily in its egg capsules.
Most neogastropods retain their eggs within some form of closed capsule (Ponder, 1973; d'Asaro, 1986; Pechenik, 1986; Smith, Black & Shepherd, 1989; Knudsen, 1994, 2000; Miloslavich, 1996a, b; Przeslawski, 2004; Pastorino, Penchaszadeh & Scarabino, 2007). With the notable exception of the free and demersal egg capsule of the volutid Adelomelon brasiliana (Penchaszadeh et al., 1999), the capsules are fixed to some substrate and moulded by a ventral pedal gland. They 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 be organized in a way that does not impede embryonic nutrition, gas exchange and excretion, and finally they should permit the hatchlings to exit (Rawlings, 1999).
South American Volutidae include 12 genera with about 30 species, but only some of their egg capsules have being investigated (De Mahieu, Penchaszadeh & Casal, 1974; Bandel, 1976; Penchaszadeh & De Mahieu, 1976; Penchaszadeh et al., 1999; Luzzatto 2006; Clench & Turner, 1970; Penchaszadeh & Miloslavich, 2001; Miloslavich et al., 2003; Bigatti et al., 2009). Odontocymbiola magellanica (Gmelin, 1791) is the only South American volutid known to deposit calcareous egg capsules (Penchaszadeh et al., 1999), but the morphology and mineralogy of the capsules, and egg-laying behaviour, have not been studied.
Odontocymbiola magellanica, the Patagonian red snail, is found from the shallow subtidal to depths of more than 800 m (Penchaszadeh & De Mahieu, 1976) and ranges through the Magellanic biogeographic province, from 35°S on the Argentine Atlantic coast (off Rio de la Plata) down to the Magallanes Strait and the Beagle Channel, reaching Chiloé Island on the Pacific coast of Chile (42°S) (Weaver & Dupont, 1970; de Castellanos & Landoni, 1992; Gallardo & Penchaszadeh, 2001; G.B., personal observation).
The current paper is part of an integrative investigation on the biology of this snail, and aims to describe egg-laying behaviour, predation on the egg capsules, the anatomy and calcium concentration of the ventral pedal gland which moulds and secretes the calcified layer, and the structure and mineral composition of the capsule itself.
MATERIAL AND METHODS
Field observations and sampling were conducted monthly, from June 2001 to September 2005 by SCUBA diving, at depths between 5 and 20 m during low tides, in Golfo Nuevo, Argentine Patagonia (42°43′S; 65°01′W). Egg capsule-laying and -moulding behaviour of Odontocymbiola magellanica was observed underwater and photographs were taken. Egg capsules at different stages of development were collected in the same area, sometimes directly from laying females. The colour, transparency and toughness of each collected egg capsule were recorded. Also, predators of egg capsules and the predated capsules were collected and identified.
Anatomy of the pedal gland
Ventral pedal glands of mature egg-laying and nonegg-laying females were observed and photographed in the field. Slices of pedal gland tissue and tissue from the periglandular sole of capsule-moulding females were fixed in either Bouin's fluid or 2.5% glutaraldehyde in filtered sea water, embedded in resin (either Leica Historesin® or Spurr resin), sectioned and stained with either haematoxylin and eosin or toluidine blue. In some other cases, they were fixed in 5% formaldehyde in seawater, embedded in paraffin and stained with von Kossa's silver nitrate procedure for histochemical Ca detection (Howard et al., 2004).
Description, and transmission and scanning electron microscopy of egg capsules
Capsular height and width were measured with callipers for 52 capsules, and the volume of their internal content was measured in a graduated cylinder to the nearest 1 ml.
Lateral fragments (3 mm wide) of recently laid egg capsules were cut and fixed for 3 h in 2.5% glutaraldehyde in 1 M phosphate buffer (pH 7.4), postfixed overnight with 1% osmium tetroxide in the same buffer, stained with 2% uranyl acetate for 45 min, dehydrated via graded ethanol and acetone, and finally embedded in Spurr's resin and sectioned in a ultramicrotome for transmission electron microscopy (TEM). For topographic orientation, 1-µm sections were stained with 1% toluidine blue in 1% potassium carbonate for 30 s. Later, silver grey sections were observed with a Philips EM 301 TEM. Also, similar 5 mm wide samples of the same capsules were fixed as described above, but they were critical-point dried, metal coated and observed under a Philips XL 30 scanning electron microscope (SEM).
Calcium determination in haemolymph, ventral pedal gland and extra-glandular mucosa
After cracking the shell, haemolymph was obtained from the heart of capsule-moulding females (haemolymph from two to three females was pooled to obtain samples of at least 1 ml). Samples of about 300 mg of the ventral pedal gland and the extra-glandular mucosa were dissected with small scissors. Samples were homogenized with 0.1 N hydrochloric acid, centrifuged at 13,000 G for 5 min, and the supernatants were kept at −80°C until the day of determinations. Total calcium was measured in 500-µl aliquots of the supernatants, which were exposed to o-cresolftalein in alkaline solution (Schwarzenbach, 1955); the resulting complexone was measured spectrophotometrically; the sensitivity of the method was 0.2 µg per tube.
Mineralogy of capsule wall
The calcareous wall of white, recently laid egg capsules was scraped, and the obtained material was air dried and powdered in a mortar, then analysed with a Siemens D 5000 diffractometer using Cu/Ni radiation. The diffractograms were compared with those of calcite and magnesium calcite standards.
Egg capsule powder was resuspended in 2.5% sodium hypochlorite, washed and dried under vacuum for 7 days at room temperature. The sample was then treated with 50% hydrochloric acid, dried, dissolved in 2% hydrochloric acid, paper filtered, diluted with an equal amount of distilled water and finally measured in triplicate with a Unicam atomic absorption spectrometer. Also, elemental analysis of hypochlorite-treated calcareous material obtained from a recently laid capsule was made by SEM (Jeol JSM 6460 LV) equipped with an EDAX PW 7757/78 X-ray energy scattering microanalyser, for standardless element quantification.
Egg capsule laying and predation in the field
Female Odontocymbiola magellanica choose flat or convex surfaces for attachment of egg capsules. The most frequently used objects were stones, bivalve shells and elasmobranch egg capsules, but they may also attach the capsules to the substrate 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 the chosen object with its foot (Fig. 1A), and it spawns a white, soft and pliable capsule (Fig. 1C, inset). It then encloses the capsule beneath its foot and adopts a stereotyped posture (Fig. 1B). The spawn is moulded and fixed to the substrate by the ventral pedal gland during this period, after which the female looses its hold and leaves behind a white, hard, round bell-shaped capsule attached to the substrate object (Fig. 1C). When taken to the laboratory, these capsules react to hydrochloric acid, producing gas bubbles, which suggests carbonate in the external cover.
The capsule-laying posture of O. magellanica is characterized by a tensing of the foot which becomes approximately conical, tilting the long axis of the shell about 45° from the horizontal plane (Fig. 1B). The siphon is only partly retracted, while the head and nuchal lobes are relaxed and flattened, exposing the eyes. However, the laying female shows an extremely low reactivity to the diver's proximity or to any disturbances. If grasped by the diver and taken out of water, it remains attached to the spawning substrate, and if forcefully detached 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 exactly the duration of egg capsule laying episodes in O. magellanica, our field observations suggest that it may take several hours.
During these field observations, predation on capsules by the green sea urchin Arbacia dufresnii and the starfish Cosmasterias lurida was observed. Predation occurs on capsules both at the beginning of development, when the calcareous cover is intact, and near the end of development, when this cover has often deteriorated (see below).
Anatomy of the ventral pedal gland
The gland located in the mid-anterior part of the sole of the 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 deposition is forcefully interrupted, the gland appears as a deep depression that corresponds exactly to the shape of the capsule which was being moulded (Fig. 2A).
Microscopically, the ventral pedal gland is composed of three distinct layers: superficial, intermediate and deep (Fig. 3A). The intermediate layer is an almost continuous space of haemocoelic cavities, muscle fibres and some connective tissue bands which separate the external epithelium (superficial layer) from apparently ‘subepithelial’ cell aggregates (deep layer).
The superficial layer is mostly composed of ciliated and goblet cells. Most ciliated cells have a slender nucleus with finely granular chromatin and no apparent nucleolus. Goblet cell nuclei may either be dark and located near the basal membrane, or similar to those of ciliated cells. In general, the secretory content of goblet cells is faintly basophilic when the secretory vacuole is basally located, but apical vacuoles are usually more intensely basophilic (Fig. 3B). On the external surface, the apices of goblet cells often occupy empty spaces in the mass of cilia. Calcium detection by von Kossa's silver-staining method showed a finely granular, dark brown deposit concentrated in the secretory vacuoles of the goblet cells (Fig. 3E). An intriguing aspect of the superficial layer is the occurrence of isolated groups of rather large oval nuclei showing heavy heterochromatic clumps; most but not all of these nuclei are located at the base of the superficial layer (Fig. 3C).
The deep layer is formed by bundles of long, bottle-shaped cells whose necks cross the intermediate layer (Figs 3A–D) and become intermingled with cells of the superficial layer, but whose cell bodies form elongated aggregates, sometimes around pseudofollicular cavities. These aggregates are perpendicular to the epithelial surface and their cells have a darkly basophilic, granular cytoplasm (Fig. 3A, D). Occasionally, a 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 surrounding epithelium of the foot by a narrow groove (Fig. 3D). The periglandular epithelium of the sole has a single layer of ciliated and goblet cells (Fig. 3D). Von Kossa staining also showed calcium in the goblet cells of the periglandular epithelium (Fig. 3F). The underlying connective tissue is traversed by muscle fibres that are perpendicular to the surface epithelium.
Calcium concentration in the ventral pedal gland and the extra-glandular mucosa
Mean calcium levels (±SE, n = 6) in both the glandular and extra-glandular sole tissue were similar (1.21 ± 0.08 and 0.97 ± 0.15 mg/ml, respectively) while the mean value (±SE, n = 6) in the haemolymph was much lower (0.070 ± 0.007 mg/ml), suggesting a similar ability of both pedal tissues to take up and concentrate calcium from the haemocoel.
Shape and size of egg capsules
Although there was a common rounded bell shape, the capsules showed a large variation in height (range, 18–41 mm; mean ± SD, 29.2 ± 6.4 mm) and width (range, 20–39 mm; mean ± SD, 30.0 ± 5.2 mm). The volume of the internal contents was also variable (range, 3.6–29.7 ml; mean ± SD, 13.2 ± 7.3 ml). Height and width of egg capsules were not correlated (r2 = 0.408; P = 0.639).
Structure and mineral composition of the egg capsule
Figure 4 is a schematic drawing of the organization of the capsule layers in recently deposited capsules. SEM showed that the capsule wall is formed by a calcareous cover (which may split into an inner and outer layer of different structure, see below) and by at least two inner membranous layers (Fig. 5A).
The calcareous layer is covered externally by a thin organic cuticle and consists of numerous spherulites (10–20 µm width; Fig. 5B) packed together in an organic matrix around septated, approximately cylindrical spaces (Fig. 5B, C). These spaces traverse the entire calcareous layer at more or less regular intervals (Fig. 5C) and their septa are mostly horizontal, though some vertical septa also occur (Fig. 5B). The septated spaces and their spherulitic walls lie on a thin layer of microfibrillar crystallites (Fig. 5A), which are in turn attached to the outer membranous layer, and represent the first calcareous material deposited by the female.
When detached from the microfibrillar calcareous layer, the underlying membranous layer has a patchy appearance (30–90 µm 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 membranous outer layer (Fig. 5D). Numerous apparent pores, about 1 µm in diameter, each surrounded by an elevated ridge, are irregularly distributed over the membrane surface (Fig. 5D). Occasionally, remnants of the inner calcareous layer are seen still attached to the membrane (Fig. 5D). We could not obtain useful preparations of the inner aspect of the membrane, mainly because of the abundant intracapsular fluid materials that remained attached to it, so that we could not establish if the apparent pores were indeed traversing the membrane.
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 associated with the development of an epibiotic community within (Fig. 5E, F). SEM observations show patches of a heterotrophic bacterial mat, diatoms and probably filamentous cyanobacteria and/or fungi (Fig. 5E, F). Eventually, the calcareous layer may disappear, leaving only the epibiotic community on the translucent membranous wall, through which crawling embryos may be seen within (Fig. 1D).
TEM showed that the membranous layers are composed of an amorphous organic matrix of low and rather uniform electron density and of numerous protein fibrils of higher electron density. The latter are more densely arranged in the outer than in the inner layers (Fig. 6A, B).
The X-ray powder diffraction pattern obtained from recently laid egg capsules showed that the calcareous material was magnesium calcite, a calcium carbonate polymorph (Fig. 7A). Atomic absorption analysis indicated that 21.1% of the Ca was substituted by Mg in the calcite lattice. EDAX standardless estimations in a similar sample (Fig. 7B) yielded a somewhat smaller figure of Mg substitution (13.4%).
The female Odontocymbiola magellanica shows low reactivity during the egg-laying process, which would appear to expose it undefended to predators. This risky parental investment may have the functional significance of creating a sealed space between the female's foot and the laying substrate. This space may serve a function similar to the ‘extrapallial’ space during shell formation (Wilbur & Saleuddin, 1983), i.e. a space in which the secreted organic matrix and a supersaturated calcium solution provide the conditions for formation of calcium carbonate crystallites. The efficiency of this capsule-secreting process is remarkable since it occurs rapidly, in hours, while shell secretion is a process taking days or weeks.
Female O. magellanica attach their egg capsules to a variety of submerged objects, but they always choose flat or convex surfaces for attaching them. It is intriguing that females of Adelomelon ancilla, which is sympatric with O. magellanica in the study area, always choose concave structures for capsule deposition. Penchaszadeh et al. (1999) recorded an illustrative example of this divergent behaviour, in which two capsules were laid on the same bivalve shell: the one of O. magellanica on the convex surface, and that of A. ancilla on the concave side.
Anatomy of the pedal gland
The distinctive shapes of neogastropod egg capsules are the result of a moulding process that occurs within a cavity formed by the ventral pedal gland, located in the sole of the female's foot (Rawlings, 1999). Histologically, the gland which moulds the capsules in O. magellanica, and which deposits the calcareous layer, consists of a ‘submerged’ epithelium similar to those found in other invertebrates (Welsch & Storch, 1976), showing a surface layer composed of ciliated and goblet cells and a deep, apparently subepithelial layer. The deep layer in O. magellanica is composed of large, elongated, bottle-shaped cells, which makes it an extreme case of a submerged epithelium. As noted by Welsch & Storch (1976), the superficial and deep layers are not separate entities (as an ‘epithelium’ and a ‘subepithelium’), 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 (Catalán, Fernández & 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 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
Neogastropods have evolved a wide variety of egg capsules, whose shape has systematic significance (Ponder, 1973) and whose primary function is thought to be protection of the developing embryo from predators and physical stresses (Rawlings, 1999).
In the case of O. magellanica, the round, bell-shaped egg capsules vary greatly in both absolute size and proportions (no correlation was found between height and width in the present study), so that these attributes may have little systematic value. The subtidal benthic deposition of these capsules protects them from physical stresses, which certainly affect the spawn of intertidal species (stresses include desiccation, osmotic stress, temperature changes and ultraviolet radiation; Rawlings, 1999). Their tough calcareous covering should make them more resistant to predators, particularly since O. magellanica shows a protracted (and direct) intracapsular development (about 2 months, Bigatti, 2005). Notably, Perron (1981) has shown for 10 Hawaiian Conus species, that capsule toughness directly correlates with developmental length. Another feature that may correlate with the long residence time of embryos within the capsule in O. magellanica is that the intracapsular fluid contains more nutrients than in other South American volutids (Bigatti, 2005).
The calcareous cover
Considering the potential protective value, it is intriguing that heavily calcified egg capsules such as those of O. magellanica and Alcithoe arabica (Ponder, 1970) appear only rarely among the Neogastropoda. The wall of some neritimorph egg capsules 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 found in the Stylommatophora (Tompa, 2005) and freshwater Architaenioglossa that deposit aerial eggs (Hayes et al., 2009). Therefore, calcareous egg capsules appear mainly as cases of convergent evolution associated with the colonization of terrestrial and freshwater habitats.
A surprising discovery is what we believe is the first record of high-magnesium calcite in molluscs. This mineral is a calcium carbonate polymorph in which Ca2+ is randomly substituted to some extent by Mg2+ in the calcite lattice; Mg substitutions over 4% (values found in O. magellanica capsules were 13.4–21.1%) are considered to be high (Ries & Blaustein, 2003). Gastropod shells are made of aragonite and to a lesser extent calcite plus aragonite, whose crystallites are embedded in an organic matrix (Watabe, 1988; Bandel, 1990). Many varied and intricate crystallite structures are formed (see Watabe, 1988, and Bandel, 1990, for references) which confer toughness and resistance to the intrinsically brittle nature of calcium carbonate.
Calcified gastropod egg capsules that have been studied are also composed of either aragonite or calcite (Bandel, 1990). An exception is the aerial egg capsules of the architaenioglossan genus Pomacea, where vaterite is found (Hall & Taylor, 1971; Meenakshi, Blackwelder & Watabe, 1974; Catalán et al., 2008). Spherulites in the calcareous capsules of O. magellanica (Figs 4, 5B, C, F) are somewhat similar to those of vaterite under the SEM, but powder X-ray diffraction analysis of the material showed a pattern like that of a magnesium calcite standard (Fig. 7).
Magnesium calcite occurs in either shells or skeletal structures of a variety of nonmolluscan groups, including sponges (Jones & Jenkins, 1970), sea urchins (Magdans & Gies, 2004), seastars (Gayathri et al., 2007), serpulid worms (Neff, 1969), crustaceans (Neues et al., 2007) and ascidians (Aizenberg et al., 2002). It is thought that magnesium substitution may impart a greater crack resistance to the calcite (Magdans & Gies, 2004).
Permeability of the egg capsule layer (which is required for gas exchange and excretion by embryos) is assured by diffusion through the membranous septated spaces and the membranous inner capsule layer, and probably by pores in the latter (Fig. 5D). Although we did not investigate the structure of the egg capsule when first spawned by the female, i.e., before being moulded by the pedal gland (Fig. 1C, inset), it is likely that some secretion or modification of the membrane occurs during the moulding process, as shown by Sullivan & Maugel (1984) in the nassariid neogastropod Ilyanassa obsoleta. Also, Price & Hunt (1973, 1974) have shown in Buccinum undatum that a ‘sclerotization’ of the membranous wall is caused by pedal gland secretions.
Seasonality of egg-laying behaviour
The egg-laying behaviour of O. magellanica is only observed during the Southern winter and spring. In general, it has been thought that water temperature and food availability are of greater importance than day length as cues for the onset and termination of reproductive processes in aquatic animals (Daly & Wilson, 1983). This view has been confirmed by observations in many caenogastropod species (e.g. Giese & Pearse, 1974; Fretter & Graham, 1994) and it has been experimentally (and quantitatively) demonstrated in an architaenioglossan caenogastropod (Albrecht, Carreño & Castro-Vazquez, 1999; Albrecht et al., 2004). The reproductive cycles of volutid snails living on the Atlantic coast of Argentina have been reported for two species that live further north (35°S) than O. magellanica: Zidona dufresnei (Giménez & Penchaszadeh, 2002) and Adelomelon brasiliana (Cledón, Arntz & Penchaszadeh, 2005) and the observed seasonal reproduction also support this general view. Notwithstanding, Bigatti, Marzinelli & Penchaszadeh (2008) have reported that the onset of capsule-laying behaviour of O. magellanica in Golfo Nuevo (42°S) occurs in July, reaches its maximum frequency in December and ceases in January (Southern summer). It seems, therefore, that the annual rhythm of oviposition and capsule-moulding behaviour in this species is entrained to increase in day length, and that this behaviour turns off in response to the small daily decreases in day length that occur after the summer solstice, even though water temperature continues to rise at that time.
Odontocymbiola magellanica is a remarkably long-lived species (up to 20 years old; Bigatti, Penchaszadeh & Cledón, 2007) reaching sexual maturity at 7–8 years old, and showing an extended oviposition season ending sharply when days become shorter (Bigatti et al., 2008). Copulatory behaviour, however, is observed throughout the year and sperm are stored in a bursa copulatrix (Bigatti et al., 2008). The slowly reacting female is exposed for hours to predators when laying an egg capsule containing 4–18 eggs and which it endows with both membranous and calcareous coverings (the latter made of magnesium calcite) and with a comparatively significant protein content for embryo nutrition (Bigatti, 2005). Furthermore, the species is prone to female masculinization (‘imposex’) and shows changes in shell morphology and female weight loss near harbours, presumably as a consequence of environmental xenobiotics (Bigatti & Penchaszadeh, 2005; Bigatti & Carranza, 2007, Bigatti et al., 2009). Therefore, O. magellanica appears as a vulnerable, though commercially exploitable snail (Bigatti & Ciocco, 2008) whose interesting morphological, behavioural and ecophysiological features should assure the continued study of its biology.
Special thanks are due to Eugenia and Victoria Zavattieri and to Oscar Wheeler for field assistance, and to Isabel Farías (IDNEU, Buenos Aires), Fabián Tricarico (MACN, Buenos Aires) and Jaime Groizard (Aluar S.A.I.C., Puerto Madryn) for help in electron microscope observations. EDAX elemental determinations were made through the courtesy of Aluar S.A.I.C. (Puerto Madryn). This work was supported by grants from FONCYT (PICTR 01869 and PICT 2008-0323) and CONICET (PIP 5301) to G.B., P.E.P. and A.C.-V., and from the National University of Cuyo (to A.C.-V.).