The Calcareous Egg Capsule of the Patagonian Neogastropod Odontocymbiola Magellanica: Morphology, Secretion and Mineralogy
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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 Patago´nico (CENPAT-CONICET). Bvd. Brown 2915, U9120ACV Puerto Madryn, Chubut, Argentina; 2Laboratorio de Fisiologı´a (IHEM-CONICET), Departamento de Morfologı´a y Fisiologı´a (FCM-UNCuyo), Casilla de Correo 33, 5500 Mendoza, Argentina; and 3Museo Argentino de Ciencias Naturales Bernardino Rivadavia (MACN-CONICET). Av. A´ngel 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 carbon- ate 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