Dataset: Urchin Fertilization Studies
Deployment: lab_SIO_Levin

Sampling and Analytical Methodology:
Spawning was induced by injecting 0.55 M KCl through the peristomal membrane. Oocytes were collected in dishes of filtered seawater (FSW, 0.22 µm). Number of adults used in each experiment ranged from 2 – 5 females and 3 – 5 males. Sperm were collected dry from the gonopores of males and placed in a small vial and kept on ice until use. A 0.1% dilution from dry sperm was made to verify sperm motility, and then preserved in formalin to count sperm densities with a hemacytometer in order to calculate the amount of diluted sperm required to attain desired sperm-egg ratios and sperm densities for each treatment. Oocytes from multiple females were mixed and egg densities determined by counting 8 x 20 µl aliquots. Eggs were added to experimental beakers at a density of 5 eggs ml^-1, and incubated in experimental conditions for 10 min before sperm were added. Dry sperm was diluted immediately before addition to experimental beakers. Following sperm addition the seawater in each replicate was gently stirred. Fertilization proceeded for 20 min and was arrested by transferring the contents of each replicate to a 1% formalin solution. The fertilization ratio, defined as the proportion of eggs fertilized, was determined by counting the frequency of occurrence of a fertilization envelope in at least 100 eggs per replicate. All experiments were carried out in the Scripps experimental aquarium facility during the spring of 2013. 500 ml glass beakers were used, and there were three replicates per treatment level.