WWW.WAS.ORG • WORLD AQUACULTURE • JUNE 2014 45 (CONTINUED ON PAGE 46) Environmental Research (CAER), West Vancouver, BC. Adult broodstock California sea cucumbers were collected sub-tidally by hand by SCUBA divers and held in indoor tanks with running, ambient, sand-filtered seawater (Fig. 2). Densities were maintained at 0.4/m2 (30 L/individual). Animals were conditioned to allow gonads to reach maturity prior to spawning by holding them at ~13 °C and feeding a prepared diet consisting of a slurry of rice bran (30 percent, by dry weight), oat bran (30 percent), wheat bran (30 percent) and Spirulina powder (10 percent) ad libitum daily. Faecal material and uneaten feed were siphoned from tanks daily before fresh food was added. Sea cucumbers were not touched or disturbed during the conditioning period. Broodstock were conditioned for eight weeks before induction of spawning was attempted. Using these procedures, successful spawning occurred throughout the year. In the laboratory, P. californicus were induced to spawn by a combination of thermal shock and exposure to suspended Spirulina, a powdered cyanobacterium1. Typically, batches of six to ten broodstock were exposed to thermal shocks 3-4° C warmer than the rearing temperature for 1 h in aerated 200-L tanks. When thermal stimulation proved ineffective, powdered Spirulina was added to tanks at 0.05 g/L. When spawning was apparent, the sex of the individuals involved was noted. To reduce the possibility of polyspermy, spawning males were removed from the tank after 7-10 min. After completion of spawning, tank water was siphoned away, and fertilized eggs were retained on a 70-μm mesh screen submerged in seawater. Eggs were rinsed three times with 1-µm filtered, UV-sterilized (UVS) seawater to remove excess sperm and any debris. Eggs retained on the screen were concentrated in a 500-mL beaker. After completely mixing eggs, 1-mL samples were collected and eggs in each sample counted using a SedgewickRafter chamber and dissecting microscope. Fertilized eggs were transferred to 20-L plastic buckets filled with 10 L of UVS seawater at approximately 50 eggs/mL. Water in rearing buckets was not exchanged and buckets were placed partially submerged inside 200-L fiberglass tanks with flow-through seawater to maintain water temperature at ~13 °C. Embryos were reared for four days until early-auricularia larvae (Fig. 3) were seen in the water column. Auricularia and subsequent larval stages were reared in 200L upwelling tanks under static conditions with UVS seawater. Constant aeration of larval cultures was provided through plastic capillaries at the bottom of tanks. Temperature during larval culture was maintained at ~13 °C. During rearing, 40-50 percent of the culture water was exchanged daily, except every fourth day, when all water was drained and larvae were retained on mesh screens ranging from 70-200 μm, according to developmental stage. At each draining, tanks were cleaned and rinsed with freshwater and UVS seawater to eliminate debris and settled phytoplankton. Larvae remaining on mesh screens were returned to culture tanks with fresh UVS seawater. During development, larvae change from the slow-moving, slipper-shaped auricularia stage to the fastmoving, oval-shaped doliolaria stage (Fig. 4). Larval density was maintained at 0.3/mL throughout the larval period because sea cucumber larvae perform well when reared at low densities (<0.3/mL) (Liu et al. 2010, Robinson 2013). Larvae were fed laboratory-cultured live microalgae (Fig. 5), which are an essential food source in the rearing of larvae of most cultured marine invertebrates. Ito and Kitamura (1997) reared larval sea cucumbers Apostichopus japonicus by feeding them the planktonic diatom Chaetoceros gracilis. Liu et al. (2010) provided a mixed algal diet (Chaetoceros muelleri, Phaeodactylum tricornutum and Dunaliella salina) for larvae of the sea cucumber A. japonicus, with ration size increasing according to developmental stage. Mixed-species algal diets typically result in better growth and survival of larvae and early juveniles of various marine invertebrates (Piña et al. 2006, RiveroRodríguez et al. 2007). However, in some instances, single-species algal diets have proven as effective as mixed-species ones, such as Dunaliella tertiolecta for larvae of the purple sea urchin Stronglycentrotus purpuratus (Azad et al. 2011). In the present study, Dunaliella tertiolecta and Isochrysis galbana were tested as single- and mixed-species diets. Algal densities were adjusted from 1×103 to 4×103 cells/mL, based on equal biovolumes of the two species; a ratio of 1:4 of D. tertiolecta LEFT, FIGURE 5. Cultured live microalgae, an essential feed for California sea cucumber larvae. RIGHT, FIGURE 6. A typical California sea cucmber pentactula larva.
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