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		We will be examining fertilization and the early stages of development in two closely-related sea urchin species. Lytechinus variegatusis from the Gulf of Mexico and Lytechinus pictusis from the Pacific coast. There is a significant difference in the mean water temperature where these species live, and at which their embryos develop. We will be comparing the rates of cleavage between these species to get an idea of the magnitude of this difference. We will also review the use of a compound microscope, examine the sperm and eggs, and look for early signs of fertilization, such as the raising of the fertilization envelope which provides a block to polyspermy (the fertilizing of a single egg by multiple sperm). 1. Induce spawning in a sea urchin (either L. variegatusor L. pictus) by injecting 1 ml 0.5M KCl solution a several sites in the soft membrane around the mouth. Rotate the urchin with your hand, place mouth down on paper towels by the sink and gently blot excess water off of the dorsal (top) surface. Within minutes, the gametes should appear: the sperm are off-white, the eggs are orange. Collect the sperm by inverting the male onto a small, dry petri dish. After several minutes, cover the sperm and put on ice. For use, dilute 3 drops of sperm in 20 ml artificial sea water (ASW, Woods Hole formula). Collect the eggs by inverting the female onto a 100 ml beaker filled with ASW at room temperature for L. variegatusand at 15-18ºC for L. pictus. Keep urchins from falling in with aluminum foil supports. Allow eggs to settle, pour off ASW and replace with fresh (at same temperature). As each of you will only inject one urchin, we will share the sperm and the eggs for observation, and we will use the L. variegatuswhich is happy at room temperature. 2. We will fertilize one batch each L. variegatusand L. pictusto allow them to begin developing. Dilute 3 drops of spermin ASW of the correct temperature. Add 10 drops to 50 ml fresh eggs and stir. NOTE THE TIME. After 5 minutes, I will check them for the presence of a fertilization envelope. After 30 minutes, you should begin to check for the first cleavage, so quickly check out your microscope. We will collect the data on the two species as a class. Please write your findings on the blackboard and initial.