BULLETIN OF MARINE SCIENCE, 51(2): 161-166,1992 FERTILIZATION SUCCESS DURING A NATURAL SPAWNING OF THE DENDROCHIROTE SEA CUCUMBER CUCUMARIA MINIATA Mary A. Sewell and Don R. Levitan ABSTRACT A natural spawning event of the dendrochirote sea cucumber Cucumaria miniata was observed in Barnfield, British Columbia, during a spring phytoplankton bloom, 18-19 March 1991. This study provides estimates of fertilization success from this spawning event and of the density of the spawning population. High percent fertilization was found in egg pellets collected soon after release from the female gonopore (if. = 87.2%; range 1-100%), and in egg pellets collected after floating to the water surface (x = 97.5%; range 68-100%). The high fertilization success seen is likely due to high population density and synchronous spawning. Marine invertebrates that broadcast-spawn thousands to millions of gametes have the potential to produce large numbers of zygotes if fertilization success is high. Recent studies, however, have suggested that fertilization success may be low unless individuals are, 1)aggregated (Pennington, 1985; Yund, 1990; Levitan, 1991; Levitan et aI., 1992), 2) synchronous in spawning (Pennington, 1985; Lev- itan, 1988; Pearse et al., 1988) and/or 3) located in low to moderate flow conditions (Pennington, 1985; Denny and Shibata, 1989; Levitan et aI., 1992). Unfortunately, the empirical evidence from these studies has relied on measurements of fertil- ization success based on induced spawning. There are no direct estimates of fertilization success of natural invertebrate spawning events [but see: Brazeau and Lasker (1990) for estimates of egg and larval production in a gorgonian; Grosberg (1991) for estimates of sperm dispersal in a clonal ascidian; and Petersen (1991) and Petersen et al. (1992) for estimates of fish fertilization success]. The present study provides the first in situ estimate of fertilization success obtained during a natural spawning event of an invertebrate. On the morning of the 18 March 1991, we noticed widespread spawning in the dendrochirote sea cucumber Cucumaria miniata in Barnfield, British Columbia. During spawning, the female releases pellets of positively buoyant green eggs, which float to the surface and break apart (McEuen, 1987). We observed eggsand eggpellets floating on or near the water surface in such numbers as to produce slicks in some locations (e.g., near the Bamfield Marine Station Dock). This widespread spawning event lasted at least 2 days and was observed in daylight hours on 18 March at sites up to 4 km apart. We used this opportunity to measure nearest-neighbor distances, population density, and the percentage of eggs fertilized soon after release and after reaching the water surface, during a natural spawning event. The results indicate that a high percent of eggs can be fertilized when population density is high and spawning is synchronous. MATERIALS AND METHODS Collection and Determination of Percent Fertilization. - Egg pellets of Cucumaria miniata were col- lected at the mouth of Barnfield Inlet, Barkley Sound, British Columbia (48°50'30"N, I25008'0"W) at noon on 18 March 1991. Pellets were scooped from the water surface or from a depth of 0.5 m and placed in separate Ziploc® bags (N = 10). Later, during a SCUBA dive at 1500 h, egg pellets were collected within a minute of release from the spawning female (N = 6 females). On the following morning (19 March), a second dive was conducted at this site to determine if the sea cucumbers were still spawning (1000 to 1100 h). Egg pellets were collected within a few minutes of release from the 161 162 BULLETIN OF MARINE SCIENCE, VOL. 51, NO.2, 1992 Table I. Fertilization success in Cucumaria miniata egg pellets collected from Barnfield Inlet on 18 and 19 March 1991. Surface = egg pellets collected within 0.5 m of the surface; Near female = egg pellets collected shortly after release from the female. A paired rank test found no significant differences between samples collected near the females and on the surface (Z = 0.637; P > 0.5) Percent fertilization 18 March 18 March 19 March 19 March Sample No. Surface Near female Surface Near female I 100.0 100.0*' 100.0 100.0*" 2 68.0 100.0 100.0*' 1.0*7 3 97.6 98.1*2 100.0 100.0 4 100.0 100.0*' 100.0 100.0 5 100.0 100.0*4 95.6 100.0*8 6 99.6 34.7 100.0 100.0*9 7 99.2 100.0 100.0 8 100.0 98.0 9 92.8 99.6 10 100.0 100.0 X 95.72 88.80 99.32 85.85 SO 10.00 26.51 1.45 37.43 • Indicates sample <250 eggs. Number of eggs sampled:" = 65; ., = 158;" = 47; •• = 100;" = 200;" = 164;" = 207;" = 36; ." = 61. female (N = 7 females); at the end of the dive, egg pellets were collected from the water surface (N = 10). Upon arrival at the laboratory, the egg pellets were transferred from the Ziploc® bags to one-liter glass jars filled with I ~m filtered sea water and left for several hours. A sample of 250 eggs from each pellet was examined under the microscope for the presence of a fertilization membrane or stages of cell division. In samples where less than 250 eggs were present, the total number of eggs was examined. In these samples egg pellets were, therefore, maintained for a period of time in a sample of sea water that may have contained dilute sperm, before being further diluted in filtered sea water. This may have resulted in an overestimation of fertilization success in egg pellets collected near the female, where nearby spawning males could have released enough sperm to cause fertilization in the collected egg/water samples within the Ziploc® bag. The degree of overestimation is probably low, since most fertilization occurs within seconds of eggs being immersed in a sperm solution (Levitan et aI., 1991). The eggs collected from the water surface, however, are not likely to be overestimates since these collections were far from males spawning 10 m below, and with rapid dilution of sperm the concen- trations would be too low to affect fertilization (Pennington, 1985; Denny and Shibata, 1989; Levitan, 1991). Therefore, the near female samples may slightly overestimate how quickly eggs become fertil- ized; however, the distantly collected egg samples provide information on the final percentage of fertilized eggs. Spatial Distribution. - To examine the spatial distribution of Cucumaria miniata. we measured density and nearest-neighbor distance. Density was estimated by randomly dropping 54, 0.25-m2 quadrats in the population of spawning sea cucumbers (1.5 to 4.0 m depth). The nearest-neighbor distance of 154 Cucumaria miniata was measured using a meter rule. Measurements were made from the center of the tentacular crown since this approximated the position of the gonopore. The sex ratio of the spawning population was not determined. RESULTS Spawning occurred during daylight hours on the third and fourth day after the new moon. The major spawning on 18 March occurred prior to 0900 h (low tide at 0735), but individuals were still spawning at 1500 h. A low level of spawning was observed between 1000 and 1100 h on 19 March (low tide at 0819). No night observations were made. Fertilization was high at all time periods, regardless of the location from which the egg pellet was collected (near female or at water surface) or the date of collection (Table 1). The mean percent of eggs fertilized soon after release was 87.2%; the percent of eggs fertilized after floating to the surface was 97.5%. In most egg SEWELL AND LEVITAN: FERTILIZATION SUCCESS IN CUCUMARIA MINIATA 163 7 6 5 4 3 2 o o 8 16 24 32 40 48 56 64 72 80 88 96 104 112 120 Density per m2 Figure I. Density of Cucumaria miniata per rn2 at the mouth of Barnfield Inlet (x = 46.0·rn-2, SD = 25.39, N = 54). pellets, nearly 100% of the eggs had been fertilized. Only 3 out of 33 pellets had fertilization below 90%. Two of these pellets were collected near the gonopore (1.0 and 34.7% fertilization) and one was collected near the water surface (68.0%, Table 1). More eggs in pellets collected near the gonopore may have been fertilized if they had been allowed to remain in the water column. The high percent fertil- ization found in the remaining egg pellets collected near the female (11 of 13 pellets with fertilization >98%, Table I) suggests that in near ideal conditions fertilization may be achieved shortly after egg pellet release. Cucumaria miniata were found at high density (x = 46.0 per m2, SD = 25.39, N = 54), with a maximum of 120 per m2 (Fig. 1). A coefficient of dispersion (CD = variance-to-mean ratio; Sokal and Rohlf, 1981) showed significant clumping (CD = 14.01, t = 72.12, df= 53, P < 0.001). The mean nearest-neighbor distance was 8.03 cm (SO = 6.22, N = 154) with a maximum distance of 50 cm (Fig. 2). A measure of aggregation (R; Vandermeer, 1981) calculated from these data did not show a significant deviation from random (R = 1.090, t = 0.013, df = 153, ns). The lack of clumping seen in the nearest-neighbor analysis is likely due to the size of the animal in relation to the distance between individuals. In many cases the animals had physical contact of their tentacles. Individuals were often seen to spawn synchronously with adjacent conspecifics. Egg pellets, once released from a female, were observed to drift back and forth 20 to 30 cm above the site of release, and thus across the tentacle crowns of adjacent, and sometimes spawning, male conspecifics before slowly floating to the surface.