World Aquacluture Magazine - September 2020

WWW.WA S.ORG • WORLD AQUACULTURE • SEP TEMBER 2020 59 ( C O N T I N U E D O N P A G E 6 0 ) UV treated) (Xing et al . 2007). Oyster shells (BOS). Crassostrea gigas shells were collected from a pond in Benth’Ostrea farm two days before the start of the experiment. Shells were broken into pieces of about 1 cm or less and 10 g of shell particles were placed into each replicate dish and filled with 100 mL FSW. Natural biofilm (NBIO). Sediment (3 kg) was obtained from a refining oyster ponds two days before the larvae experiment. Sediment was manually homogenized with a spoon and spread in a 3-cm thick layer on a plastic tray. Two-layered lens tissue was placed on the sediment surface for 12 h to imitate the natural tidal and light cycle (Eaton and Moss 1966). The upper layer of the lens tissue was removed and the contents washed to harvest diatoms, which were transferred into 500 mL of FSW (Fig. 3). The mixture (100 mL) was inoculated into each replicate and held for 24 h at 15 C, a 14 h light, 10 h dark cycle, and a light intensity of approximately 100 μmol/m 2 sec. Filtered seawater (FSW). Filtered seawater (100 mL) was used as a negative control to estimate the percentage of larvae undergoing spontaneous metamorphosis. Metamorphosis Assay Purple sea urchin larvae were reared in the Benth’Ostrea Pond aquaculture farm in the Breton marshes near Bouin, Vendee, France. Before the experiment, pre-competent larvae were transferred to the Laboratoire Mer Molécules Santé in Nantes, France and held at 1 larva/mL in an aerated 5-L glass reactor ball until competence was achieved. Larvae were considered competent when 75 percent had developed a rudiment that was equal to or greater than the size of the stomach (Kelly et al . 2000). Competent, eight-arm purple sea urchin larvae (N=750) at 15 days post-fertilization (Fig. 4) were used in the FIGURE 6. Metamorphosis rate (mean %± SD) of P. lividus larvae after 11 days exposure to treatments of NIT, NIT + A, BOS, NBIO and FSW. first part of the experiment. Competent larvae were stocked into 80-mm Pyrex Corning crystallizing dishes with 100 mL filtered seawater (FSW). Thirty larvae were pipetted into each dish and were maintained in a natural photoperiod regime at 20 C. All replicates were fed 1-2 mL of Dunaliella tertiolecta daily. Metamorphosis rate was evaluated using a stereomicroscope after 72 h of exposure to treatments and then checked daily until the eleventh day of exposure, until at least one treatment reached 90 percent metamorphosis rate. Larvae were classified as metamorphosed once they presented a circular appearance, with tubed feet, spines, and were attached to the surface (Fig. 5). Metamorphosis rate (percent) was calculated as metamorphosed post-larvae / total number of larvae × 100. After the first 72 h of exposure, only the NBIO treatment had some metamorphosed larvae (3 percent). On the eleventh day of exposure, the metamorphosis rate was 67 percent in the NIT treatment, 49 percent in the NIT + A treatment and 41 percent in the NBIO treatment. The metamorphosis rate was 13 percent in the negative control (FSW) and no larvae metamorphosed in the BOS treatment. NIT, NIT + A, and NBIO had a significantly greater metamorphosis rate than FSW and no differences were observed between NIT, NIT + A and NBIO (Fig. 6). Growth and Survival of Post-Larvae Growth and survival of post-larvae was evaluated using the FIGURE 4. Eight-armed competent purple sea urchin larvae. FIGURE 5. Metamorphosed P. lividus with tube feet and spines. FIGURE 3. Homogenizing sediments in preparation of natural biofilm (left), removal of lens tissue (center), harvesting of diatoms in 500 mL of filtered seawater (right).