Cah. Biol. Mar. (2006) 47 : 243-251 Embryonic development of the tropical bivalve Tivela mactroides (Born, 1778) (Veneridae: subfamily Meretricinae): a SEM study Thomas SILBERFELD and Olivier GROS* UMR 7138 Systématique-Adaptation-Evolution, équipe Symbiose Université des Antilles et de la Guyane, U.F.R des Sciences Exactes et Naturelles. Département de Biologie. 97159 Pointe-à-Pitre Cedex, Guadeloupe (France). *Corresponding author: Tel 590 48 92 13, Fax: 590 48 92 19, E-mail [email protected] Abstract: The embryonic development of Tivela mactroides, from fertilization to straight-hinge veliger D-stage larva occurs in 18 hours at 25°C. Scanning electronic observations show that morphogenetic processes result in a gastrula with two depressions 4 hours after fertilization (T0 + 4h). Two hours later, one depression, located at the animal pole, develops into an open cave, the floor of which becomes the shell field located below the lower face of the prototrochal pad. The invagination located at the vegetal pole features the blastopore. At T0 + 6h, the late gastrula has differentiated into a typi- cal motile trochophore with a shell field synthetizing the organic part of the shell. At T0 + 8h, the shell field, located between the prototroch and the telotroch, appears as a saddle-shaped region with a wrinkled surface extending on both sides of the embryo, establishing bilateral symmetry. At T0 + 12h, the prototroch slides toward the anterior region by outgrowth of the shell material. At T0 + 18h, the prodissoconch I formation is completed and the D-stage larvae possess a calcified shell. At this stage of development, the functional velum is composed of four bands of cilia. Our observations seem to confirm the interpretation of shell differentiation proposed recently by Mouëza et al. (2006) for bivalves even if Transmission Electronic observations will be necessary to validate definitively such a model in the Meretricinae sub-family. Resumé : Développement embryonnaire du bivalve tropical Tivela mactroides (Born, 1778) (Veneridae : Meretricinae) : une étude en microscopie électronique à balayage. Le développement embryonnaire de Tivela mactroides, de la féconda- tion au stade larve D se déroule en 18 heures à 25°C. Les observations en microscopie électronique à balayage montrent que les processus morphogénétiques aboutissent 4 heures après fécondation (T0 + 4h) à une gastrula pourvue de deux dépressions : celle située au pôle animal se développe en une large cavité dont seul le plancher, situé sous le bourrelet prototrochal, correspond à la région coquillière, l’invagination du pôle végétatif représentant le blastopore. À T0 + 6h, la gastrula s’est différenciée en une trochophore mobile avec une région coquillière synthétisant la coquille embryonnaire. À T0 + 8h, la région coquillière, d’aspect fripé, prend une forme en haltère qui, en se développant de chaque côté de l’embryon, établit la symétrie bilatérale. À T0 + 12h, la prototroche est déplacée vers la région antérieure suite à l’expan- sion de la coquille. À T0 + 18h, la formation de la prodissoconque I est terminée et la larve D possède une coquille calcifiée. Cette larve véligère possède un velum fonctionnel constitué de quatre bandes de cils. Nos observations semblent confirmer Reçu le 4 octobre 2005 ; accepté après révision le 5 juillet 2006. Received 4 October 2005; accepted in revised form 5 July 2006. 244 EMBRYONIC DEVELOPMENT OF T. MACTROIDES le modèle de différentiation de la coquille proposé par Mouëza et al. (2006) pour les bivalves, cependant des observations en microscopie électronique seraient nécessaires pour valider définitivement un tel modèle dans la sous-famille des Meretricinae. Keywords: Mollusca l Bivalvia l Ontogenesis l SEM Introduction Materials and methods Embryonic, larval and post-larval development of bivalves has long been studied with a view to elaborate an aquacul- Individual collection ture of species with economic interest, to identify the Tivela mactroides BORN 1778 is a large clam (up to 45 mm systematic location of species at larval planktonic stages, or in shell length) belonging to the family Veneridae (subfami- to understand ontogenesis processes occurring specifically ly Meretricinae), distributed from West Indies to Brazil in this invertebrate embranchment. In Eulamellibranchia, (Warmke & Abbott, 1962; Abbott, 1974). It lives shallowly larval development has been first described in a number of burrowed in the sandy bottoms of the Guadeloupe coast species with as much details as possible using light where it is widely collected and consumed under the microscopy (Loosanoff & Davis, 1963; Loosanoff et al., T. mac- 1966; Frenkiel & Mouëza, 1979; Sastry, 1979). More vernacular name of “Chaudron”. Adult specimens of recently, some papers were published about the embryonic troides were collected by hand in June 2004 in shallow development of bivalves with scanning electron sandy bottoms of “La Source” beach in Guadeloupe (Fig. 1). microscopy (SEM) analysis, such as those on pectinids Spawning and fertilization (Hodgson & Burke, 1988; Bellolio et al., 1993), lucinids (Gros et al., 1997 & 1999), on a venerid (Mouëza et al., Adults of T. mactroides can spawn throughout the year in 1999), and on a nuculid (Zardus & Morse, 1998). Few the field depending to environmental stresses (personal papers have combined TEM and SEM analysis as those of observation). For each spawning assay, 20-30 freshly Eyster & Morse (1984) on Spisula solidissima, Casse et al. collected individuals were used. Specimens were carefully (1998) on Pecten maximus, Zardus & Morse (1998) on cleaned by scrubbing their shell, and placed in a 50-litre Acila castrensis, and Mouëza et al. (2006) on the venerid tank filled with seawater, until the onset of spawning. Chione cancellata. These last authors have even proposed a Various stimulations as osmotic stress (individuals were new model to explain the shell formation and differentia- placed in fresh water up to 15 minutes), one-hour exunda- tion in bivalves (Mouëza et al., 2006). In order to confirm tion on a sunny place, and serotonin injections were used to such model, we have checked embryonic development and induce spawning. Once spawning was completed, adults shell-field differentiation in a new investigated bivalve sub- were taken out. The density of the embryos was adjusted to family. 30.000 per litre. The water was then softly bubbled direct- Moreover, the embryonic development of T. mactroides ly in the spawning tank, in order to avoid sedimentation of will allow an ontogenetic comparison with the other species eggs and embryos and also hypoxia due to the high oxygen of venerid bivalves already described. Indeed, Ansell consumption by the embryos themselves. Development (1962) pointed that the study of developmental stages of was proceeded at room temperature, i.e. 25°C, at a salinity members of the family Veneridae was particularly relevant, of 36 ± 1 which is typical for their natural habitat. No while this family is considered as a crown group within the antibiotics were used and all seawater used for fertilization Eulamellibranchia Molluscs (Giribet & Wheeler, 2002). and rearing was filtered through a 5-µm Millipore cartridge The purpose of the present paper is (i) to describe the filter. whole embryonic development in a Meretricinid species, (ii) to compare patterns of embryonic development in Sampling and fixation tropical venerids, and (iii) to investigate selected aspects of ontogenesis in this family in order to confirm the new shell To study the successive embryonic stages of T. mactroides, differentiation model recently suggested in bivalves by 500 mL of seawater containing 4-5 thousands of embryos Mouëza et al. (2006). were sampled on a 26-µm mesh nylon screen every hour T. SILBERFELD, O. GROS 245 Figure 1. Map of Guadeloupe showing the location of the site of bivalve collection: La Source. Figure 1. Carte de la Guadeloupe indiquant la localisation du site de la Source. throughout the entire embryonic development (i.e. ~ 20 with gold (Sputter Coater SC500, Biorad) before observa- hours). The embryos were then placed in a small basket for tion in a SEM Hitachi S-2500 at a 20 kV accelerating a safe handling and transferred into a fixative solution of tension. 2% glutaraldehyde in filtered seawater at 4°C for at least 2 hours before dehydration. Results The first D-shaped larval stages have required a previous specific treatment, in order to avoid the retraction of velum inside the calcified valves when glutaraldehyde is added. Spawn induction The simplest way to do that was to put larvae in a small volume of seawater at -20 °C for 5 minutes, so that most of Adult individuals of Tivela mactroides spawned in the the larvae could not retract their velum (frozen anaes- hatchery throughout the year as observed in their natural thetized-like) when pouring the fixative solution. environment (personal observations). In bivalves, spawning can be induced by applying a stress of various SEM preparation nature (for review, see Mouëza et al., 1999). According to Matsutani & Nomura (1982) and Gibbons & Castagna After the fixation, embryos were twice rinsed in 0.1 M cac- (1984), a chemical stress has been applied, by injection of codylate buffer adjusted to pH 7.2 and 1000 mOsM, then a 2 mM serotonin solution in 0.22 µm filtered seawater in dehydrated through a graded acetone series, before drying the visceral mass. But T. mactroides did not seem to be sus- by CO at critical point in a Biorad apparatus (Polaron, 2 ceptible to this chemical stimulus. Therefore, other kinds of Critical Point Drier). Then samples were sputter-coated stress were tested such as osmotic stress by putting the 246 EMBRYONIC DEVELOPMENT OF T. MACTROIDES clams into freshwater at room temperature for 15 minutes about 50-µm long. The middle piece appears made of four followed, or not, by a one-hour exundation step on a sunny spherical mitochondria (Fig. 3). Fertilization normally place. This last method has been successful. We usually occurs and no polyspermy was observed in the several observed a spawning of male and female gametes within 30 batches obtained during this study.