We will be examining the efect of varying the chromosome number on the early stages of development in Lytechinus variegatus. We will compare normal development to that of parthenogenotes (artifically activated eggs) and of eggs fertilized 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/brown. 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. variegatus. 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. Work in whatever groups you are comfortable in(2-3). Fertilize one batch L. variegatuseggs (use about one third of a batch of eggs diluted to 50 ml) to allow them to begin developing. Dilute 3 drops of spermin ASW of the correct temperature. Add 20 drops to 50 ml fresh eggs and stir. NOTE THE TIME. After 5 minutes,check them for the presence of a fertilization envelope. 3. If your eggs fertilize reasonably well, inhibit the establishment of the slow block to polyspermy by adding 5 ml 4 mg/ml soybean trypsin inhibitor to a second batch of eggs in 45 ml. Dilute fresh sperm, and fertilize eggs. Check after fertilization, and for abnormalities at cleavage. 4. Parthenogenesis is the activation of an egg without sperm contact. Activate a third group of eggs by hypertonic shock. Allow remaining eggs to settle. Resuspend in hypertonic sea water (ASW with 30 g NaCl per liter), allow to settle for 20 min. Wash eggs by pouring off Hypertonic sea water and resuspending in normal ASW. Check for fertilization membrane elevation. After 10 min. wash again and check. Alternative method: Divide eggs into four 50 ml tubes, allow to settle. Resespend in 50 ml ASW (tubes 1 and 2) and 50 ml ASW with 5 mM NH4Cl added to cause increase in intracellular pH (tubes 3 and 4). Add 0.5 ml calcium ionophore A23187 (1 mg/ml in DMSO) to tubes 2 and 4, and 0.5 ml DMSO to tubes 1 and 3 (as controls). Check for fertilization envelope elevation and for cleavage. 5. Observe and follow the best one or two batches of eggs activated under the 3 conditions through cleavage divisions to hatching and gastrulation (hopefully). Document any abnormalities that you observe.