LABORATORY REARING OF THE LARVAE OF THE MAHOGANY DATE MUSSEL LITHOPHAGA BISULCATA JOHN L. CULLINEYl Duke University Marine Laboratory, Beaufort, North Carolina 28516 ABSTRACT Although adults of Lithophaga bisulcata did not spawn readily in the laboratory, fertilized eggs were obtained after six adventitious spawnings of captive adults. Larvae, developing from these eggs, were reared through pelagic stages in the laboratory. Eggs were 64p, in diameter, the gastrula 66p, long, and the trochophore 67p. X 57p,. Shelled stages in- creased in length from 92p, to 400p, during pelagic development, and a broad umbo appeared when larvae were between l40p, and 160p. long. The hinge structure of the larval shell consisted of a series of taxodont teeth at either end of the hinge line. Shelled larva did not possss an apical flagellum. Trochophores and straight-hinge larvae appeared positively phototactic. Pediveligers developed large eyespots and were able to delay metamorphosis for at least one month. Larvae resembled larvae of Modiolus demissus much more than those of Mytilus edulis, but had a broader umbo and more rounded ventral margin. Also, in older larvae of L. bisulcata, the umbo appeared to shift anteriorly from the midline. INTRODUCTION The mahogany date mussel, Lithophaga bisulcata Orbigny, is a small, bullet-shaped member of the family Mytilidae. It bores into calcium carbonate material in shallow marine and estuarine waters. Adults are commonly 1 to 1.5 inches long. The range for the species is from North Carolina to the Gulf of Mexico and the West Indies (Abbot, 1954). More recently, Turner & Boss (1962) have reported the species from the island of St. Helena in the eastern Atlantic. These authors also have cited depth records for L. bisu/cata from the intertidal zone to 79 fathoms. Occurring in tropical and subtropical waters, the genus Lithophaga is often associated with coral reefs, where it may be one of the most abundant rock-destroying organisms in the reef environment (Gardiner, 1931; Otter, 1937). Near Cape Lookout, North Carolina, L. bisulcata commonly bores into heads of the coral OCUlifUl arbuscula, where at times it is the dominant species of the infaunal community (McCloskey, 1967). L. bisulcata is also partly estuarine, occurring in the Newport River, North Carolina, to at least 3 miles inland from Beaufort Inlet; here, it bores into large, empty pelecypod valves, chiefly MercefUlria mercenaria. There is a virtually complete lack of knowledge concerning the life 1 Present address: Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts 02138. 592 Bulletin of Marine Science [21 (2) history of any species of Lithophaga. The only reference to the larvae of a date mussel is the statement of MacGinitie (1935) that larvae of L. plumula in California metamorphosed to the bivalve stage in 10 days. He gave no description of the larvae or of their development. The purpose of this paper is to describe and illustrate the larvae of L. bisu/cata so that they may be identified in the plankton. Furthermore, the established feasibility of rearing large numbers of these larvae to meta- morphosis provides the opportunity to study their initial penetration and destruction of calcium carbonate material. This may prove of interest to students of reef ecology and evolution, and also perhaps to those in- vestigating biological penetration of hard calcareous substrates. MATERIALS AND METHODS Adult mussels were collected in Bogue Sound, North Carolina, in July 1968. They were removed from their burrows and maintained in 1 to 2 liters of filtered sea water by feeding concentrated suspensions of unicellular algae (Isochrysis galbana and Platymonas sp.). Water and food were renewed daily. All spawning and fertilization occurred naturally. Many attempts were made to induce spawning, using such techniques as rapid warming and cooling, adding sperm from a ripe male, and pricking the posterior adductor muscle with a pin. However, no spawning response occurred within 24 hours following any of these procedures. Whenever spawning occurred, larvae were isolated from their parents at the earliest swimming stage and reared using the basic techniques of Loosanoff & Davis (1963). Larvae were reared in I-liter cultures at a constant temperature of 250 ± I°C and a salinity of 30;~o± 2%0, and were fed chiefly I. galbana. Water and food were renewed daily. Initial food- cell densities daily varied between 5 X 104 and 1 X IOu cells per cc. Initial larval population densities varied between 10 individuals per cc and 20 per cc, becoming less than 5 per cc as the larvae reached the pediveliger stage. Larvae were measured with a calibrated ocular micrometer. Most of the terminology relating to larval shell structure is that discussed by Chanley (1965, 1967). OBSERVATIONS AND DrSCUSSION Spawning.-Adult date mussels with ripe gonads did not spawn readily in the laboratory. The techniques of raising the water temperature and adding sperm from a ripe male worked neither as separate procedures, nor when combined. L. bisulcata seems to behave as some other common mytilids in that its spawning cannot be easily or quickly induced. Loosanoff & Davis (1963) reported considerable difficulties in their attempts to spawn Mytilus edu/is and Modiolus demissus in the laboratory. Fertilized eggs were obtained from spawnings of six different groups of 1971] CulLiney: Larvae of Lithophaga bisulcata 593 TABLE 1 SUMMARY OF MAJOR FEATURES OF LARVAL DEVELOPMENT IN Lithophaga bisulcata Stage or distinctive Development feature Size time Unfertilized egg 64p. Earliest swimming stage 66p. 5-6 hours (ciliated gastrula) Trochophore 67p. long; 57p. 9-12 hours greatest diameter Straight-hinge veliger earliest shell development 20-24 hours Straight-hinge veliger 92p. X 67p.; hinge-line 66p. 36 hours Development of umbo > 1401' to 1601' long 7-9 days Mature larva, development 300p. to 400p. long 26 days of foot, eyespots present adults. In all cases, spawning occurred between 2 and 6 days after the animals were removed from their burrows. No clear correlation between spawning and a sudden increase of the water temperature was noted. In one case, mass spawning occurred while the animals were gently agitated on a lab shaker. In all cases, a few unfertilized eggs appeared 1 to 2 days prior to mass spawning of fertilized eggs. A few individuals with broken shells were included in all the spawning groups, and these individuals may have participated significantly in the spawning. The technique of pricking the posterior adductor muscle with a pin, successfully used by Loosanoff & Davis (1963) to stimulate spawning in M. edulis, did not seem to work for L. bisulcata. Conceivably, spawning in Lithophaga could be governed by photo- period. This possibility has not been investigated. Daily rhythms in amplitude and frequency of shell movement in L. lithophaga, a Mediter- ranean species, are regulated by photoperiod (Salanki, 1966). The possible effect of photoperiod on spawning in other mytilids might also be in- vestigated. Larval Development.-Development time, sizes of various larval stages and the appearance of prominent features are summarized in Table 1. Ripe unfertilized eggs of L. bisulcata were medium to dark brown, with a large, more lightly colored nucleus. They were quite uniform in size, averaging 64p. in diameter. Five to 6 hours after fertilization, at tempera- tures between 25° and 30°C, the embryos developed into the earliest swimming form, a brown, ciliated gastrula, approximately 66p. long. By 9 to 12 hours after fertilization, the embryos were dark brown molluscan trochophores, measuring 67fI. long and 57fI. across the greatest diameter. 594 Bulletin of Marine Science [21 (2) FIGURE 1. Lithophaga bisulcata, early straight-hinge larva, 99p..long X 77p. high. _ t • FIGURE 2. Lithophaga bisulcata, late straight-hinge larva, 132p.. long X llOp. high. 1971] Culliney: Larvae of Lithophaga bisulcata 595 FIGURE 3. Hinge structure of young larvae of Lithophaga bisulcata. Length of hinge-line in center larva is 64p,; largest hinge teeth are 4 to 5p, wide. FIGURE 4. Larva of Lithophaga bisulcata, early umbo stage, 160p, long X 143p, high. 596 Bulletin of Marine Science [21 (2) FIGURE 5. Group of larvae of Lithophaga bisulcata, showing development of umbo. Largest larva is 266p. long X 240p. high. Trochophores and, to a lesser extent, earliest shelled stages seemed to be positively phototactic. In culture vessels taken from the dark, early larvae seemed at first fairly evenly distributed between the surface and bottom. But under overhead lights, within 1 to 2 minutes, masses of larvae ap- peared at the surface. Within a day after the larvae developed shells, this phenomenon no longer occurred, and larval distribution in the culture vessels was generally uniform. The larvae first developed shells 20 to 24 hours after fertilization. By 36 hours after fertilization, prior to being fed, average measurements of straight-hinge larvae were 92p. long and 67p. high, with a hinge-line 66p. in length. A straight hinge-line was present in larvae up to approximately 130p.long (Figs. 1 and 2). A number of interlocking hinge teeth were present in the young larval shell. Teeth were quite prominent at either end of the hinge-line, the largest measuring approximately 5p. in width. Toward the middle of the hinge- 1971] Culliney: Larvae of Lithophaga bisulcata 597 I r L_ FIGURE 6. Larvae of Lithophaga bisulcata, late umbo stage, showing shift in position of umbo anteriorly. Larvae are approximately 3301-' to 3601-' long. line the teeth became smaller, and they seemed nearly to disappear over the central portion (Fig. 3). The umbo began to appear in larvae over 140fL long. By the time larval length exceeded 160fL, the umbo had become a broad, prominent structure. It then appeared more and more nipplelike as the larval shell grew longer beyond the edges of the umbo (Figs. 4 and 5). The fully developed umbo FIGURE7.