SOME ASPECTS OF SPAENING BEHAVIOR AND DEVELOPDENT IN LITTORINA PLANAXIS AND LITTORINA SCUTULATA Kent Imai May 30, 1964 SOME ASPECTS OF SPAMNING BENAVIOR AND DEVELOPMENT IN LITTORINA PLANAXIS AND LITTORINA SCUTULATA Heretofore, observations on the spawning and development of the internally fertilizing intertidal snails, L. planaxis and L. scutulata, have been limited to studies conducted by Hewatt (1938) and by Glynn (1963). Recent observations at Hopkins Marine Station on the central California coast further elucidate the reproductive biology of these littorines. These studies, conducted in the lab¬ oratory and in the field in the period April 21 to May 29, 1964, shed some light on spauning behavior and suggest relationships between development and ecological conditions pertaining to these two species. L. planaxis and L. scutulata are oviparous, and females of both species have been observed to lay eggs in the field and in the laboratory. The egg mass is extruded from beneath the right side of the mantle cavity in the region of the genital pore. Females have been seen in various postures during the egg laying process - alone or when paired with a male, while crawling or quiescent - and in all cases have been attached to their substrate by the foot. In one instance, a female discharged eggs while a male was attempting copulation. In the field, egg laying snails were observed on vertical and horizontal rock surfaces, at the air- water interfaces of tide and splash pools, and beneath the water surfaces of these pools. Field observations coupled with laboratory efforts suggest that there are at least three factors which may induce egg laying. These are 1) changes in light intensity, 2) physical disturbances, and 3) moisture shock. In the field, the greatest number of egg masses were located in tide pools and splash pools during the hours corresponding to daun and dusk. In two separate instances, fifteen (15) and twenty-four (24) egg masses were layed at 6:00 A.M. and 7:00 A.M., respectively. On three other occasions, one (1), five (5), and thirty- five (35) egg masses were discharged between the hours of 8:00 P.M. and - 2 . 8:15 P.M. (Fig. la) In the laboratory, efforts to induce egg laying by means of changes in light intensity met with success on only one occssion. In this instance, five females were placed in a finger-boul of sea water and were kept in a dark room for a period of fifteen (15) hours. One hour after exposure to sunlight, one female discharged an egg mass. The effect of physical disturbance on laying behavior was suggested by the fact that in several instances, females extruded their egg masses on dry surfaces at various hours during the day and night just after having been brought to the laboratory from the field. Also, agitation of the water in which snails were kept was followed by the extrusion of egg masses in two instances, after periods of fifteen (15) minutes and tuo (2) hours. Moisture shock appears to be the most effective stimulus to egg laying. In the field, the only recorded instances of egg laying on vertical or horizontal rock surfaces isolated from pools were noted during or just after subjection to wave action (Table I). Figure lb suggests a very slight correlation between incidence of egg laying and subjection of the snails to wave action from the rising tide. In addition, forty (40) of the eighty-three (83) egg masses observed in the field were found on the morning of May 6. Twenty-five (25) of these egg masses were located in high splash pools which were not reached by wave action but which were affected by a rainstorm which occurred during the night. In the laboratory, moisture shock was utilized as a reliable stimulus to egg laying. On numerous occasions, eggs were obtained from females which had been sprayed with sea water from a tap. Upon subjection to this moisture shock, egg were extruded after periods ranging from five (5) minutes to just over two (2) hours. Individual females were never observed to discharge more than one egg mass after copulation. Many females were kept in the laboratory after having deposited a single spawn. These individuals were subjected periodically to sudden changes n eaetttes P S Iln e H TABLE I. ECG MASSES LAID IN THE FTELD TIME 4 OF MASSES SPECIES DATE 2015 A. 35 plar 5-12 L. scutulata B. 24 L. planaxi 5-6 0700 C. 15 L. planaxis 5-6 0600 L. planaxis 5-11 2015 E. 2010 L. scutulata5-13 F. L. scutulata5-6 0100 G. 2 L. planaxis 5-19 1800 POOL TENA 14.5 C 17.5 C 10.5- 11.0 C 9.5 C 14-150 8,5 C SALINITY .O19N. .O17N. .019— evap. .019 .018 COMTNTS Eggs found in a series of pools ranging in height from open sea to 8 ft; 6" deep. 20 batches floating, 5 at bottom, 10 scattered be¬ tween pools and in lower pools. Both species in a¬ bundance at edges & on bot- tom of pools. Eggs found floating (16) and at bottom (8) of a pool 6" deep and 20 ft above mean low water. Eggs found floating in pool 8 ft above mean low water. Eggs laid in lab- planaxis taken from horizontal rock face- 2 ft above mean low water Scutulata observed laying egg mass just below water surface in pool+8 ft; 6" deep Scutulata observed laying egg mass in pool plus 28 t; 8 " deep Eags laid on vertical rock surface plus 12 ft during splashing 152 3 in light conditions, agitation, and moisture shock, but even after two weeks, further deposition of spaun was not exhibited. The egg laying behavior of these littorines, both in the field and in the lab¬ oratory, was conspicuous for its synchronous nature. That is, egg masses fron groups of females in close proximity to each other on the substrate, or living in the same rock pool, were often deposited over a period of time not exceeding one hour. This was established by noting that eggs from the several masses were at the same stage of development at any one time. In the laboratory, the deposition of eggs by one individual seemed to affect other individuals in the same finger-bol, causing them to spawn. This apparent synchrony of egg laying suggests the possi¬ bility of some sort of ectohormonal activity. A more likely possibility is that, after copulation, the female will delay the egg laying process until such time as favorable conditions prevail. Spawning stimuli are likely to affect many individuals occupying similar environments, thus causing the phenomenon of synchrony. Keeping females in isolation after copulation indicated that the egg laying process may be delayed for as long as three (3) days. When freshly laid, the L. planaxis egg mass contains an average of 1200 - 1300 eggs, although spaun masses with as few as 700 and as many as 2000 eggs have been counted. The composition of the spawn is similar to that described for L. neritoides by Fretter and Graham (1962). The eggs are enclosed individually in saucer shaped capsules (one egg per capsule). The capsules, in turn, are held toge¬ ther in a mucus matrix, and are oriented such that they are stacked one on top of the other (Figs 2a, 2b). The developing eggs average 77 microns in diameter (range - 70 to 84 microns), are spherical in shape, and of a pinkish hue. Each egg, with a surrounding store of albumen which is retained by a membrane, is enclosed in a raised bulge which accomodates the developing embryo in the center of a transparent capsule averaging 349 microns in diameter (range - 340 to 364 microns). (Dimensions Lgare 2: Lplasssis, sager f develgginent 21 al- albamen ei- cilia ep capsule e developir embogo f - foat m- membranc ma- mucus matx op opercular sh- shell V- yelam V- viscerat hump . . imm om 2. Heshlg laidegg mass 6. freshig laid egg mess, deldt -35 mm 2. Ensapsalgted 10m 4. Devslging srnrga. Li dej Lone. kerg 10m . 10 mm e. Deusloping embege, 3p tr 2 oniautes tejgre tathirg f. Veliget C -4. are averages of twenty measurements.) (Fig. 2c) The work of Ciselin (1964, unpublished) suggests that the capsule may be a mucus-like substance composed of galactogen and some protein. The entire spaun mass is arranged in a spiralling configuration composed of from one to two rings. Each L. scutulata spaun contains from two to three times as many eggs as the individual spawn of L. planaxis. The capsules are arranged in a similar fashion, except that there are from three to five rings in the spiral configuration. In all other respects, the egg masses of L. planaxis and L. scutulata are identical. This casts some doubt on previous observations that each transparent capsule contained as many as eight developing snails (Glynn, 1963), for none of the L. scutulats capsules observed in this study contained more than one egg. In the field and in the laboratory, the freshly laid egg masses were often observed floating on the surface of the water. Gaseous bubbles held by surface tension to the surface of the mucus matrix were found to be responsible for this buoyancy. These bubbles, presumably originating from the gas within the mantle cavity of the snail, were not observed on the matrix surfaces of submerged spauns. In any case, the mucus matrix began to break up within one day, at which time the liberated capsules sank and continued development below the surface of the water. The sequence of development was followed in the laboratory for L. planaxis embryos. Rates of development were compared for eggs kept at 25 degrees C., at room temperature (15.8 - 21.5 degrees C.) and at sea water temperature (11.0 - 14.0 degrees C.). (Fig. 3) Discrepancies in the rates of development for eggs at room temperature and for eggs at sea vater temperature are attributed to fluc¬ tuations in the range of temperatures obtaining under these two conditions. Like most molluscs, the L. planaxis egg is holoblastic and cleavage is of spiral type. The first two divisions are meridional and result in four blastomeres of approximately equal size. The third division is horizontal and results 15 155 156 C . in an eight celled stage of four mieromeres and four macromeres. Drauings of subsequent stages are presented in Figure 2. Hatching occurs from tuo (2) to eight-and-three-quarters (8 3/4) days after spawning. The nearly incessant ciliary movement of the encapsulated embryo just before hatching seems to assist the larva in its struggle to escape confinement. The larvae vacate their capsules through the ruptured wall of the raised bulge in the center of the capsule. The studies by Linke (1933) on L. littorea, as cited by Fretter and Graham (1962), indicate that osmotic pressure within the capsule may be partly responsible for the break in the wall. L. Planaxis and L. scutulata hatch as free-swimming post-torsional veliger larvae. There are no grossly obvious characteristics which help to distinguish one from the other. The most obvious morphological structures visible under the micro- scope are a bilobed velum ringed with cilia, a shell, an operculum and a slightly developed foot. Thus, Hewatt's identification of trochophores belonging to the species L. planaxis appears to be erroneous (Hewatt, 1938). The pelagic veligers swim about by means of ciliary movement. The natural positions when swimming is with the bilobed velum and foot above and the shell below. Slight agitation of the water seems to have no effect upon their swimming movements, which appear to be random. If, while swimming, the larvae encounter any objects or surfaces, they immediately cease ciliary movement and sink to the bottom. They resume swimming momentarily. If a veliger is resting on the bottom, it can be made to retract completely into its shell by tapping on the microscope stage. The veligers of L. planaxis and L. scutulata have been kept alive for up to eight days after hatching without indications of settling. The foot of the larva, even after eight days of pelagic life, appears unsuited as yet to the assumption of a benthic existence. Presumably, the pelagic life of these two species lasts considerably longer than eight days. 151 75 Attempts to locate developing embryos in the field have met with little success. In the tide pools and splash pools, eggs were found in all stages of development up to the presence of moving cilia. This corresponds to just over two days of development at room temperature. Most of the eggs recovered from these pools had begun to lose their characteristic pinkish hue, and looked to be inviable. This suggests that eggs which remain in the higher rock pools are subject to ecological pressures which prevent their complete development. Such pressures might include increased salinity due to evaporation, or the presence of predators which could conceivably feed on the developing eggs as they lay quiescent on the pool bottoms. Surface plankton collected from the open sea a few feet from the rocky shore yielded a single veliger which resembled the larva of L. planaxis or L. scutulata. On the basis of these field observations, it appears that the eggs of L. planaxis and L. scutulata must somehow attain the open sea. It is possible that these species may be induced to spaun during periods of wave action, when the possibility that the eggs will reach the sea is greatest. After a period of pelagic development, the veligers may settle on rocks in the low intertidal and assume the crawling mode of life. 15 LITERATURE CITED FRETTER, V. & A. GRAHAM 1962. British Prosobranch Molluscs. Ray Society, London. GLYNN, P. W. 1963. Thesis on the Endocladia - Balanus Association. Hopkins Marine Station, Pacific Grove, California. HEWATT, W. G. 1938. Notes on the breeding seasons of the rocky beach fauna of Monterey Bay, California. Proceedings of the Calif. Academy of Sciences, Fourth Series, volume 23; 283 - 288.