<I>Cucumaria Miniata</I>

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 fertilization 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 collected 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 fertilized; 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

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 fertilization 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.

DISCUSSION In this study we observed high fertilization success in nearly all Cucumaria miniata egg pellets sampled. This may be the result of a number of mechanisms that increase the probability of fertilization in this sea cucumber. These mechanisms include spawning at low slack tide, small distances between nearest neigh- bors, large population size, spawning behavior, egg buoyancy, and spawning synchrony. 164 BULLETIN OF MARINE SCIENCE, VOL. 51, NO.2, 1992

>. 20 u =~ = 15 ~C' I. r.. 10

0 0 5 10 15 20 25 30 35 40 45 50 N-N Distance (cm) Figure 2. Nearest-neighbor distance (N-N distance, crn) in Cucumaria miniata at the mouth of Barnfield Inlet (x = 8.03 em, SD = 6.22, N = 154).

Spawning at low tide has been postulated as a mechanism to increase the concentration of gametes by reducing the volume of water into which gametes are released (McDowall, 1969). In Asterias forbesi, an inverse relationship was found between water depth and fertilization success (Levitan and Rumrill, unpub1. data). Spawning at slack tide also decreases the rate of gamete dilution, due to reduced water flow. Both experimental evidence and predictions from models suggest that fertilization success is inversely related to water flow (Pennington, 1985; Denny and Shibata, 1989; Levitan et aI., 1992). During slack tide, Yund (1990) noted fertilization success to be over 80% up to 3 m from a spawning male hydroid colony. McEuen (1988) has also conducted in situ observations indicating that, in high current areas in the San Juan archipelago, large populations of dendrochirotes release their gametes during periods of slack water. Experimental evidence also suggests an inverse relationship between distance from a spawning male and fertilization success (Pennington, 1985; Denny and Shibata, 1989; Levitan, 1991; Yund, 1990). Pennington (1985) noted fertilization success to be approximately 90% when eggs were located only 10 cm away from a spawning male sea urchin. Similar results have been found by Levitan (1991) and Yund (1990). Brazeau and Lasker (1990) have suggested that the low ratio of larval to egg production found in a gorgonian population may be due to poor fertilization success resulting from large distances between con specifics (mean distance between each female colony and the nearest male = 74 cm). Our estimate of average nearest-neighbor distances was only 8 cm; 95% of the sea cucumbers were within 20 cm of a con specific. Population size and density of spawning animals have also been found to be important to fertilization success (Levitan, 1991; Levitan et aI., 1992). In an experimental study conducted in the same location as this spawning event, fertilization success of sea urchins averaged 30% and up to 80% at densities of only 4· m -2 with 8 spawning males (Levitan et aI., 1992). Our estimate of population density in Cucumaria miniata was 46· m -2 with a population size in the thousands. Spawning behavior may also playa role in increasing fertilization success in Cucumaria miniata. During spawning, Cucumaria miniata stretches the anterior third of the body out from the rocks and up into the water column, keeping the tentacles in a motionless state (McEuen, 1988; pers. observations). Egg pellets SEWELL AND LEVITAN: FERTILIZATION SUCCESS IN CUCUMARIA MIN1ATA 165 and sperm are thus released into the water column without disturbance due to feeding activities of the tentacles. The buoyant nature ofthe eggs, combined with the extension of the spawning adult, places the gametes into the water column, facilitating gamete dispersal and fertilization. A similar spawning behavior in the sea cucumber Bohadschia marmorata, where the tentacles are shielded by the tentacular crown during spawning, has been suggested to allow for unimpeded dispersal of gametes (Hendler and Meyer, 1982). By washing back and forth over the gonopores of close conspecifics while slowly rising to the surface, the eggs sample a large volume of water, increasing the chance of passing through a sperm plume. Babcock et al. (1986) have suggested that the buoyant nature of the eggs of Great Barrier Reef corals may also increase the rate of fertilization by concentrating gametes at the surface. This is, however, depen- dent on sperm buoyancy which has not been investigated in Cucumaria. Adult spawning behavior and egg buoyancy may also decrease the chance of capture by filter feeders and other benthic predators. The motionless state of adult tentacles may limit mortality due to cannibalism. Free from the benthos, mortality would be restricted to pelagic predators. Cucumaria miniata eggs are toxic to fish (Shimek, 1987), but the larvae may still be vulnerable to jellyfish and other pelagic predators during their short planktonic period (8-13 days; McEuen, 1987). The apparent synchrony of spawning, suggested by the observation of spawning individuals, the enormous number of egg pellets seen in the water, and the exposed outstretched appearance of all adults in the population, would also increase the chance of fertilization. Synchronous spawning of individuals has been reported in many taxa, with the timing of these events related to a multitude of factors such as temperature, phases of the moon, tidal pressure changes, salinity, and nutrient/food availability (see review in Giese and Kanatani, 1987). This spawning event occurred during a spring phytoplankton bloom consisting mainly of diatoms (pers. observations). Similar spawning events of Cucumaria miniata during spring phytoplankton blooms have been reported by McEuen (1988). We also observed species spawning simultaneously with Cucumaria miniata: the congener Cucumaria piperat'a (2 adjacent males; K. Durante, pers. comm.) and the asteroid Henricia spp. (2 males). Neither species was in close proximity to spawning female conspecifics. A similar multi-species spawning event of echinoderms at low density was observed in Barkley Sound by Pearse et al. (1988). Spawning individuals were within 1m of each other in only two cases, and gametes were rapidly diluted (Pearse et aI., 1988). Our results suggest that under near ideal conditions of flow, depth, population structure, and spawning synchrony, high fertilization success can be achieved. Yet even under these conditions, one egg pellet collected from the water surface had relatively low fertilization success (68%). Assuming high in situ fertilization rates are followed by normal embryonic and larval development, these data suggest that free-spawning of gametes can be an effective mechanism for producing large numbers of offspring (fecundity in Cucumaria miniata up to 1.318 x 105, McEuen, 1987), but only under fairly restricted population structures and environmental conditions. However, we still need information from spawning events in a wide range of species, under a variety of population sizes and environmental conditions. Collecting these data will depend on the serendipity of observing such events.

ACKNOWLEDGMENTS

We thank K. Cook, K. Durante, and C. Levitan for assistance in the field; K. Durante, C. Levitan, and T. Rawlings provided comments on the manuscript. This study was supported by a University of Alberta graduate assistantship and a Sigurd Tveit Memorial Scholarship to MAS, a Barnfield Marine Station Research Fellowship to DRL and a NSERC of Canada grant to F-S. Chiao 166 BULLETINOFMARINESCIENCE.VOL.51. NO.2. 1992

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DATEACCEPTED: November 21, 1991.

ADDRESS: Zoology Dept .. University oj Alberta. Edmonton, Alberta, Canada T6G 2E9 and Bamfield Marine Station, Bamfield. British Columbia. Canada VOR 1BO. Reprint requests to: M. Sewel/, Zoology Dept .. University oj Alberta, Edmonton, Alberta, Canada. T6G 2E9.