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		Procedure 1. Gametes from healthy adult Lytechinus variegates were collected and fertilized following Gamete collection and Fertilization protocol. a. Gamete collection i. 3 mL of 0.5 M KCl was injected to induce spawning. ii. Using Artificial Sea Water (ASW), eggs were collected in a beaker, washed and stored at room temperature. Sperm were stored at 4 °C in a small test tube after collection in a dry Petri dish.   b. Fertilization i. 5 mL of eggs were allowed to settle in a test tube after being transferred from the beaker. All but 2 mL of ASW were pipetted out. ii. Three drops of sperm were diluted in 5 mL of ASW (diluted sperm) five minutes before use because viability in water is short-term. iii. Diluted sperm were observed with a compound microscope to ensure motility. iv. Eggs were fertilized with 2 drops of diluted sperm. v. ASW was added until the tube was 90% full 5 minutes post-fertilization. vi. A depression slide was used to view 2-3 drops of eggs 10 minutes post-fertilization. vii. Fertilization envelopes were observed.   2. Fertilized eggs were divided into three Petri dishes, control, Treatment A, and Treatment B. 3. ASW was added to control treatment, labeled, and moved to a secure location to develop. 4. After the fertilized eggs in Treatment A reached the two cell stage (70-90 minutes post-fertilization), excess ASW liquid was removed and eggs were re-suspended in calcium-free seawater, photograph taken. 5. The fertilized eggs in Treatment B were allowed to develop until they reached the blastula stage (about 12 hours post-fertilization). 6. Excess ASW removed from Treatment B and replaced with calcium-free seawater. 7. Embryos continued to develop and photos were taken throughout a 24 hour period and compared to the control embryos which developed only in ASW.   Download Handout