EMBRYONIC AND LARVAL DEVELOPMENT OF ENSIS ARCUATUS (JEFFREYS, 1865) (BIVALVIA: PHARIDAE) FIZ DA COSTA, SUSANA DARRIBA AND DOROTEA MARTI´NEZ-PATIN˜O Centro de Investigacio´ns Marin˜as, Consellerı´a de Pesca e Asuntos Marı´timos, Xunta de Galicia, Apdo. 94, 27700 Ribadeo, Lugo, Spain (Received 5 December 2006; accepted 19 November 2007) ABSTRACT The razor clam Ensis arcuatus (Jeffreys, 1865) is distributed from Norway to Spain and along the British coast, where it lives buried in sand in low intertidal and subtidal areas. This work is the first study to research the embryology and larval development of this species of razor clam, using light and scanning electron microscopy. A new method, consisting of changing water levels using tide simulations with brief Downloaded from https://academic.oup.com/mollus/article/74/2/103/1161011 by guest on 23 September 2021 dry periods, was developed to induce spawning in this species. The blastula was the first motile stage and in the gastrula stage the vitelline coat was lost. The shell field appeared in the late gastrula. The trocho- phore developed by about 19 h post-fertilization (hpf) (198C). At 30 hpf the D-shaped larva showed a developed digestive system consisting of a mouth, a foregut, a digestive gland followed by an intestine and an anus. Larvae spontaneously settled after 20 days at a length of 378 mm. INTRODUCTION following families: Mytilidae (Redfearn, Chanley & Chanley, 1986; Fuller & Lutz, 1989; Bellolio, Toledo & Dupre´, 1996; Ensis arcuatus (Jeffreys, 1865) is the most abundant species of Hanyu et al., 2001), Ostreidae (Le Pennec & Coatanea, 1985; Pharidae in Spain. Its commercial value has increased consider- Hu et al., 1993; Chaparro, Thompson & Emerson, 1999), Pecti- ably in recent years. Ensis arcuatus lives buried in sand in low nidae (Cragg, 1985, 1989; Bellolio, Lohrmann & Dupre´, 1993; intertidal and subtidal zones from Norway to Spain and it is Benninger, Dwiono & Le Pennec, 1994; Casse, Devauchelle & also found along the British coast (Hayward & Ryland, 1998). Le Pennec, 1998) and Veneridae (Goodsell et al., 1992; The accurate identification of bivalve larvae and post-larvae Moue¨za, Gros & Frenkiel, 1999, 2006). However, embryonic in planktonic and benthic samples is essential to both basic development cannot be ascertained in detail using light and applied research efforts in estuarine and open coastal microscopy, and much of the research undertaken on bivalve marine environments (Gustafson & Lutz, 1992). Identification developmental patterns lacks descriptions of the early stages of planktonic larvae is crucial for investigating larval dispersion, (Verdonk & Van den Biggelaar, 1983). settlement events, levels of recruitment and growth rates of The aim of this study was to describe the developmental infaunal bivalves (Gribben & Hay, 2003). Positive larval identi- pattern of the razor clam E. arcuatus, from hatching to metamor- fication can only be verified by direct techniques such as larval phosis, using light and scanning electron microscopy. Knowl- culture (Chanley & Andrews, 1971). The use of shell morpho- edge of E. arcuatus embryology and larval development is metrics such as length, height and thickness has proved useful necessary to obtain characters for the planktonic and benthic in identifying larvae (Gribben & Hay, 2003). However, some identification of samples and to improve larval rearing in closely related species present similar valve structures, making hatcheries. species identification difficult (Le Pennec, 1980; Fuller & Lutz, 1989), and more so if several species of the same family live in the same area. Some studies on bivalve larval development MATERIAL AND METHODS have proposed that the internal structure and morphology of the valves and soft parts of the larvae show specific differences Larval culture (Elston, 1980; Cragg, 1985, 1989; Hodgson & Burke, 1988). Fifty individuals greater than 100 mm in length were collected Moreover, characters that can be easily detected by light by skin-diving from a natural bed in a subtidal zone of Cies microscopy can also contribute to larval identification. In Islands in Rı´a de Vigo (Galicia, NW Spain). At Centro de Cul- recent years, alternative methods showing accuracy, specificity tivos de Ribadeo-CIMA, razor clams were held in row bars tied and speed have been used for field studies. Molecular methods individually with rubber bands in open circuit. such as DNA-based (Andre´et al., 1999; Hare, Palumbi & Razor clams were taken from the holding tank for spawning Butman, 2000) and immunological methods (Lorenzo, Fuentes and placed in groups of 25 individuals into 200-l tanks. Four & Gonza´lez-Ferna´ndez, 2003; Lorenzo et al., 2005) show con- different stimulations along with a control were used to induce siderable promise for the identification of bivalve larvae. These spawning: thermal shock with addition of microalgae and methods do not contribute toward the improvement of hatchery gonad extracts; changing water level by simulating tides, with larval culture, however, because larval morphology cannot be brief dry periods; stripping gonads; and UV-treated seawater ascertained nor abnormal larvae identified. flow. The specimens responding to the stimulus were separated Larval development has been described in several species of into individual receptacles for the release of sperm or eggs, Eulamellibranchia, providing basic knowledge for aquaculture thus avoiding self-fertilization. After fertilization, the eggs were and for identification of larval stages in the plankton (Loosanoff sieved to eliminate excess sperm. & Davies, 1963; Loosanoff, Davies & Chanley, 1966). With Gametes were then transferred to 500-l larval culture tanks scanning electron microscopy (SEM) different aspects of larvae with aerated water at a temperature of 19 + 18C. Water was and post-larvae have been studied in several species of the changed every 2 days using 1 mm filtered, UV-sterilized sea- water. D-stage veliger larvae were fed daily with Tetraselmis suecica, Isochrysis galbana, Pavlova lutheri and Chaetoceros calcitrans Correspondence: F. da Costa; e-mail: fi[email protected] at 40 cells/ml as initial ration. Journal of Molluscan Studies (2008) 74: 103–109. Advance Access Publication: 27 February 2008 doi:10.1093/mollus/eym051 # The Author 2008. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved. F. DA COSTA ET AL. Electron microscopy length, was produced by epiboly and the vitelline coat no longer surrounded it (Fig. 1H). Originally located at the Throughout embryonic development, in each stage at least 100 vegetal pole, the blastopore was displaced to the ventral side of individuals were collected and photographed using an optical the embryo during gastrulation. In the late gastrula the circular ¼ microscope. Eggs and larval stages were measured (n 100). margin of the blastopore presented some cilia, and on the dorsal Samples were fixed in 2.5% glutaraldehyde in 0.1 M sodium side a groove called the ‘shell field’ was located opposite the blas- cacodylate buffer solution. After rinsing in cacodylate buffer, topore (Fig. 2A). embryos were dehydrated in an ascending series of ethanol After 19 h (T0 þ 19 h) the late gastrula had differentiated into washes and critical point-dried using CO2. Embryos were then a typically pyriform trochophore, measuring approximately sputter-coated with gold before observation in a Philips XL30 60 mm in length, swimming actively in the water column SEM. (Fig. 2B). The trochophore had a crown of motile cilia, called Confirmation of prodissoconch structure in Ensis arcuatus was the prototroch, which divided the trochophore into two areas. achieved using live 5-day-old larvae by cryofocusing, in which The area posterior to the prototroch was occupied by the blasto- live larvae were frozen in liquid nitrogen to avoid the alteration pore, which differentiated into the mouth on the ventral side and of larval structures after SEM preparation procedures. Live the shell field, which was surrounded by a ciliary tuft, on the Downloaded from https://academic.oup.com/mollus/article/74/2/103/1161011 by guest on 23 September 2021 larvae were placed in a cavity of a cylindrical aluminum plate dorsal side. The newly secreted shell material spread out, and and the plate was then submerged in liquid nitrogen for 5 min. folded into right and left segments connected by a thicker During cryotransfer to the SEM, larvae were shielded in a band, representing the first hinge. Behind it, in the postero- Bal-Tec VCT100 cryoholder. A JEOL JSM-6700F microscope dorsal region, a tuft of cilia dorsal to the anus represented the tel- was used to study larval morphology. The cryoholder was otroch. The anterior area constituted an apical plate with the / inserted into the microscope and the larvae larval chamber long ciliary tuft of the apical flagellum at its centre, formed by was pumped for about 10 min to remove water vapour. Speci- a group of associated cilia with a length of 12 mm. 2 8 mens were studied at 160 C. Typical straight-hinged D-shaped larvae developed from a trochophore by 30 hpf and these were 109.36 + 10.03 mm long RESULTS (Fig. 2C). The prodissoconch I progressively enclosed the soft body parts, but the time of calcification was not observed. Adult individuals of Ensis arcuatus spawned in the hatchery from From straight-hinged larvae to metamorphosis, a wrinkled pel- January until April. Neither thermal shock, stripping gonads, licle was seen over the valves in samples prepared for SEM. Con- nor UV-treated seawater flow were effective as spawning firmation of prodissoconch structure in E. arcuatus using live methods. Changing water levels by simulating tides was the 5-day-old larvae by cryofocusing showed prodissoconch I and only successful method, the first individuals spawning 2 h after II (Fig. 2D). The surface of prodissoconch I presented a punc- the beginning of induction. In our experience, six females out tate pattern and prodissoconch II commarginal growth lines. of a batch of 25 individuals spawned, each female releasing The larval velum, oval-shaped with a few rows of cilia measur- approximately 1 million eggs.