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 67p. X 57p,. Shelled stages in- creased in length from 92p, to 400p, during pelagic development, and a broad 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. 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 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 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. Mature larva of Lithophaga bisulcata, 409fL long by approximately 370# high. Focus is on apparent gill rudiment posterior to foot. 598 Bulletin at Marine Science [21(2)

FIGURE 8. Group of mature larvae of Lithophaga bisulcata, 350p. to 400p. long. Reflected light was used, to show the eyespots. is the knobby type of Chanley (1967). The shape of the umbo of larvae over 160,...long made them look much more like larvae of Modiolus demissus than Mytilus edu/is. (Compare Figures 7 and 10 of Loosanoff et al. [1966] with Figure 5 in this paper.) However, the umbo of larval L. bisulcata was more prominent than that of Modiolus, and its position changed relative to that of Modiolus as the pediveliger stage was neared. In such maturing larvae of Lithophaga, shell growth posteriorly seemed to exceed growth in the anterior direction. Thus, the umbo was shifted anteriorly from the original, central position (Fig. 6). In Modiolus, the umbo remains in a nearly central position through metamorphosis. Differential growth in length and height of the larval shell did not occur at least up to a length of approximately 300,.... Length remained constantly 20,...to 25,... greater than height from the straight-hinge stage through the late umbo stage. However, at lengths over 300p., accurate shell measurements became difficult due to increasing roundness of the larvae, which caused them to roll into different positions during measurement. By the 26th day after fertilization, most larvae were between 300,... and 400,... long and possessed a large, apparently fully developed foot (Fig. 7). However, none of the larvae were seen to crawl at this time. The smallest larva in which a developing foot was seen measured approxi- mately 290,...long. This individual also appeared to possess a developing 1971] Culliney: Larvae of Lithophaga bisuIcata 599 gill rudiment. An apical flagellum was never evident in shelled larvae. A pair of prominent eyespots appeared in late-stage (Fig. 8). These were well developed in larvae over 300fL in length. They were discovered only fortuitously by observing the larvae with reflected light. Lying beside the opaque mass of the digestive gland, the eyespots were not easily seen with transmitted light, and it is not known at what stage they first appeared. Delay of Metamorphosis.-Observations of late-stage larvae indicate a capacity of this species to delay metamorphosis for long periods. Twenty- six days after fertilization all the cultures of Lithophaga were discontinued. However, approximately 100 larvae, 300fL to 400fL long and 26 days old, were accidentally left in filtered sea water with no added food at room temperature (22° to 25°C). When these were noticed again 29 days later, most of the larvae were still alive, and none had metamorphosed. There had been no gradual loss of the velum, as described by Bayne (1965) for Mytilus edulis. Rather, the larvae were able to swim normally. Agita- tion of the culture vessel seemed to stimulate swimming. Feeding these larvae with lsochrysis also stimulated swimming and crawling. This was the first time crawling had been observed in larvae of L. bisulcata. Crawling consisted of a smooth gliding motion, using the extended foot. The mechanism of crawling appeared to be entirely ciliary; no muscular locomotory movements were noticed. Within 1 to 2 days after feeding, the velum became noticeably reduced, and increased development of the gills was seen in some individuals. Further observations of metamorphosis and postlarval growth were not made, due to infestations of the young dissoconchs with epiphytic growths (unidentified fungi and/or blue-green algae). The capacity of larvae of Lithophaga bisulcata to delay metamorphosis may account for the extensive geographical distribution of this species. Its presence On both sides of the Atlantic suggests that long-distance trans- port of larvae may occur in the equatorial current systems. Such "long distance larvae," originating from shallow-water, adult populations on either side of the Atlantic, are becoming well known in many different groups of animals, especially gastropods (Scheltema, 1968). Delay of metamorphosis in other species of Lithophaga might also be investigated, particularly L. aristata, which ranges widely on both sides of the Atlantic as well as in the eastern Pacific (Turner & Boss, 1962). In addition to larval transport, the dispersal of Lithophaga may occur due to its presence in fouling communities on ship bottoms (Turner & Boss, 1962). These authors mentioned having received a number of young L. bisulcata taken from a buoy. This habitat is either unusual for date mussels, or else they do not grow nearly as large and conspicuous as they do in calcareous material. Many interesting experiments might be done on metamorphosis in 600 Bulletin of Marine Science [21 (2) Lithophaga, and comparisons should be made with nonboring mytilids. The ability of a species to delay metamorphosis may be functionally cor- related with the degree of specificity or rarity of its preferred substrate. Nonborers such as Mytilus edulis perhaps encounter suitable substrate as pediveliger larvae much more often than does Lithophaga. Examples of pelagic larvae having highly specific and often rare requirements for metamorphosis are the shipworms (Teredinidae) and various commensal organisms. However, the effects of specific bacterial films shown to be important in settling of Brachidontes lineatus (spelling corrected from the original paper of Kiseleva, 1966) must be further investigated. Also, such factors as gregarious behavior, high concentration of food and high inten- sity of light, which stimulate settling of larvae of Ostrea edulis (Bayne, 1969), should be carefully considered.

ACKNOWLEDGMENTS This research was done at the Duke University Marine Laboratory, with the support of a U. S. Fish and Wildlife Service Graduate Education Grant (No. 14-17-0007-401 [G]). I thank Mr. Paul Chanley and Dr. C. G. Bookhout for their constructive criticisms of the manuscript.

SUM ARlO

CRfA EN LABORATORIO DE LA LARVA DEL MEJILL6N Lithophaga bisulcata Mejillones adultos, Lithophaga bisulcata, fueron colectados durante el mes de julio en Bogue Sound, North Carolina y desovaron en ellaboratorio de 2 a 6 dias despues que fueron removidos de sus escondites. Las larvas se desarrollaron desde los huevos fertilizados a travcs del estado de troc6fora hasta el estado de concha con charnela recta en 20 a 24 horas. Las larvas alimentadas principalmente con lsochrysis galbana a1canzaron el estado de pediveliger en aproximadamente 26 dias a 25° ± 1°C de temperatura y 30%0 ± 2%0 de salinidad. El diametro del huevo promedio fue 64ft. La longitud de las larvas can concha aument6 de 90ft a 400ft durante el desarrollo pelagico. La linea de la charnela tenia 66ft de largo. Un umbo ancho y redondo apareci6 primero a las 140ft de longitud y se volvi6 mas prominente a medida que la longitud de la concha excedia 160ft. La longitud fue constantemente de 20ft a 25ft mas grande que la altura, par 10 menos hasta una longitud de 300ft. Los troc6foros y las larvas j6venes con charne1a recta parecian positiva- mente fototacticas. La estructura de la charnela de la concha larval de los juveniles estaba formada par una serie de dientes de la charnela entrelazados que median 1971] Culliney: Larvae of Lithophaga bisulcata 601 alrededor de 5Jk de ancho en los extremos de la linea de la charnela y decredan en tamafio hacia el centro. Las larvas no tenlan flagelo apical. Las pedivellgeras desarrollaron un par de grandes manchas ocu]ares. Las larvas de L. bisulcata en los ultimos estados del umbo eran distintas de las larvas de Modiolus demissus y Mytilus edulis en la estructura y posicion del umbo y forma del borde ventral de la concha. Las larvas pediveIlgeras fueron capaces de demorar su metamorfosis por 10 menos un mes en 22° a 25°C.

LITERATURE CITED ABBOTT, R. T. 1954. American Seashells. D. Van Nostrand Co., Princeton, New Jersey, 541 pp. BAYNE, B. L. 1965. Growth and delay of metamorphosis of larvae of Mytilus edulis. Ophelia,2: 1-47. 1969. The gregarious behavior of the larvae of Ostrea edulis L. at settle- ment. J. mar. bioI. Ass. U.K., 49: 327-356. CHANLEY, P. E. 1965. Larval development of a boring clam, Barnea truncata. Chesapeake Sci., 6(3): 162-166. 1967. Aids for identification of bivalve larvae of Virginia. M.S. thesis, College of William and Mary, 64 pp. GARDINER, J. S. 1931. Coral reefs and atolls. MacMillan and Co., London, 181 pp. KISELEVA, G. A. 1966. Factors stimulating metamorphosis of a lamellibranch, Brachyodontes lineatus Gmelin. [In Russian, English summary.] Zool. Zhur., 45: 1571-1573. LOOSANOFF, V. L. AND H. C. DAVIS 1963. Rearing of bivalve mollusks. Pp. 1-136 in Russell, F. S. (Ed.), Advances in marine biology. Volume 1. Academic Press, London, 410 pp. LOOSANOFF, V. L., H. C. DAVIS, AND P. E. CHANLEY 1966. Dimensions and shapes of larvae of some marine bivalve mollusks. Malacologia, 4(2): 351-435. MCCLOSKEY, L. R. ]967. Community dynamics of the fauna associated with Ow/ina arbuscula Verrill (Coelenterata, Scleractinia). Ph.D. dissertation, Duke Uni- versity, 187 pp. MACGINITIE, G. E. 1935. Ecological aspects of a California marine estuary. Am. MidI. Nat., 16: 629-765. OTTER, G. W. 1937. Rock destroying organisms in relation to coral reefs. Sci. Rep. Gt Barrier Reef Exped., 1(12): 323-352. SALANKI, J. 1966. Daily activity rhythm of two Mediterranean lamellibranchs (Pecten 602 Bulletin of Marine Science [21 (2) jacobaeus and Lithophaga lithophaga), regulated by light-dark period. Ann. Inst. BioI. Hung. Acad. Sci., Tihany, 33: 135-142. SCHEL TEMA, R. S. 1968. Dispersal of larvae by equatorial ocean currents and its importance to the zoogeography of shoal-water tropical species. Nature, Lond., 217(5134): 1159-1162. TURNER, R. D. AND K. J. Boss 1962. The genus Lithophaga in the western Atlantic. Johnsonia, 4(41): 81-116.