World Aquaculture March 2019

WWW.WA S.ORG • WORLD AQUACULTURE • MARCH 2019 47 ( C O N T I N U E D O N P A G E 4 8 ) Institute for the Sea and Atmosphere (IPMA) in Olhão recently started scientific work to develop techniques for breeding, larval development and post-larvae, growth and survival of the purple sea urchin, with special emphasis on the development of cultivation protocols to promote larval survival. Broodstock Collection and Conditioning Wild broodstock were collected manually in rocky areas during low tide. Urchins were transported in boxes at low density to avoid excessive contact between animals. Boxes were covered to protect urchins from direct sunlight and to keep themmoist, and then quickly transported to EPPO. At EPPO, animals were placed in fiberglass tanks in an open system with filtered water (Fig. 1). The cultivation water temperature followed natural seasonal variation; however, cooling during the warmer months was needed so that temperature would not exceed 22-23 C. Aeration was relatively strong because these animals occupy turbulent habitats, heavily influenced by wave action. The provided diet was primarily green seaweed ( Ulva spp.) and this was readily accepted by urchins the day after individuals were collected. Other species of macroalgae were tested, such as Saccorhiza polyschides (Laminaria), Cystoseira usneoides , Codium sp . and Asparagopsis armata ) that were collected in the intertidal zone on the southwest rocky coast of Portugal. However, purple sea urchins did not have any interest in consuming any of these macroalgae. Another advantage of using Ulva spp. as the main food for sea urchin production was the availability of vast quantities of this seaweed in earthen ponds at EPPO. Spawning Induction Echinoidea class members are dioecious (individuals are one sex) animals. However, sea urchins do not exhibit sexual dimorphism and so it was possible to differentiate sexes only during the emission of gametes. There are several approaches to induce spawning referenced in the literature but the most efficient method was osmotic shock (Gago et al . 2009). To provoke this osmotic shock, 1 mL of 0.5 M potassium chloride was injected into the celomic space through the peristomial membrane (Fig. 2). After injection, sea urchins were stirred manually for 1-2 min to facilitate diffusion of the solution injected into the celomic cavity and then placed inverted on top of a glass to carry out gamete collection (Fig. 3). If the sea urchin was sexually mature, gamete emission would occur in a few minutes. The oocytes were orange, while the sperm was whitish. Sperm was collected using a micropipette. Oocytes were diluted in sterile sea water for counting. When the total number of oocytes was determined, sperm could be added. Dilution was important because an imbalance between gametes could compromise fertilization success by the occurrence of polyspermy. There is still work needed in this area, but in a very simplistic way, the use of a ratio of 500 male gametes for every oocyte can be used with good results. Fertilization rate was assessed through microscopic observation of samples two hours after mixing gametes. The presence of eggs with a fertilization membrane (Fig. 4) meant successful fertilization (McBride 2005). Hatching and Larval Culture Fertilized eggs were then transferred to a tank for hatching. Sufficient water renewal to maintain good water quality for the entire column is necessary, a flow equivalent to 10 percent water exchange per hour is advised. The water inlet should be preferably located on the tank bottom and the outlet at the surface should be provided with a 55-µm plankton net to prevent egg loss. Regular monitoring is required to avoid an overflow. Aeration was carried out by a small air diffuser and crystal tubes of small diameter without a diffuser to promote a greater turbulence. Embryonic development (Fig. 5) was followed by regular observation with a stereoscopic microscope. After hatching, and to decrease handling, the larvae (planktonic phase) could be kept in the same tank but all residual organic material, such as capsules and non-fertilized eggs, must be removed. Larval cultivation at EPPO was carried out at a controlled temperature of 21 to 23C. Dissolved oxygen was monitored by FIGURE 3. Emission of purple sea urchin gametes after induction with 0.5 M KCl (Photo: João Araújo). FIGURE 4. Schematic illustration of the difference between non- fertilized and fertilized eggs in sea urchin.