connected to a thermostat slowly increased the 

 temperature to 27.0° ± 0.5°C. After 30-60 min at 

 this temperature, we extracted gametes by syr- 

 inge (as above) for use in fertilization exper- 

 iments. To protect gametes from large pressure 

 changes during gonad extractions, we maintained 

 a gentle and constant negative pressure in the 

 syringe; gametes drawn abruptly into the syringe 

 were discarded. Only extractions which entered 

 the syringe as dense white cords were used; more 

 diffuse or cloudy extractions were also discarded. 

 Samples of gonad extractions and all seawater and 

 glassware used were stored at 27.0° ± 0.5°C. 



Gamete Concentrations and Fertilization 



Extractions from the gonads of 3-5 males were 

 transferred to and lightly agitated in a Klett- 

 Summerson sample tube containing 5-8 ml of 

 seawater. Using a Klett-Summerson colorimeter 

 with a green (#54) filter, we measured light 

 diffusion through diluted extractions. We then 

 diluted subsamples, placed them in a hemacy- 

 tometer, and counted the number of sperm. Com- 

 parisons were then made between the Klett read- 

 ing (K) and the actual sperm counts. 



Gonad extractions from 3-5 females were 

 similarly pooled, transferred to a Nytex screen 

 (36-;um mesh), and rinsed with seawater to remove 

 small debris and reduce the possibility that some 

 component of the eggs (released from any ova 

 broken during extraction) would cause sperm to 

 agglutinate (Galtsoff 1964). We then rinsed the 

 cleaned eggs into a 250-ml beaker containing 20-50 

 ml of seawater, counted samples of eggs using a 

 dissection microscope, diluted samples with 

 seawater until reaching the desired concentration, 

 and maintained the egg-seawater suspension at 

 27.0° ± 0.5°C. We discarded eggs remaining in 

 seawater for more than 1 h to reduce the pos- 

 sibility of sperm agglutination resulting from 

 secretions. 



Using a Pasteur pipette (45 drops of seawa- 

 ter/ml), we transferred various sperm concentra- 

 tions (Table 1) to numbered Syracuse watch 

 glasses, then with an automatic pipette added 0.2 

 ml of egg-seawater suspension containing 100 ± 4 

 eggs. The pH of 7.8 ± 0.1 was in the range recom- 

 mended by Humphrey (1950). After introduction 

 of the egg-seawater suspension, we added 7 ml of 

 seawater at different time intervals (flooding 

 time. Table 2) to dilute the sperm concentrations 

 and to reduce the possibility of polyspermy. The 



Table 1. -Range and mean of percent fertilization (%Z) and 

 percent of larvae developing to the D-shape stage (%D) resulting 

 from different sperm concentrations combined with fresh eggs 

 (100 ± 4/0.2 ml) of Crassostrea gigas.^ 



'Seawater of salinity 25%o and pH 7.8 it 0.1; temperature 27° 

 ± 0.5"C; gametes diluted with 7 ml of seawater at 10 min post- 

 fertilization; 5 repetitions per sperm concentration. 



Mean percent larvae losses = (%Z- minus %Dj). 



'h 



^Cl = Compatibility index 



/l -(%Z -) 



X ^o^. 



Table 2.-The range and mean of percent fertilization (%Z) and 

 percent of larvae developing to the D-shaped stage (%D) 

 obtained by different flooding times i after combining the 

 gametes of Crassost rea gigan.'^ 



'Flooding time = time (min) between the combination of ga- 

 metes and the addition of 7 ml of seawater to the gamete mixture. 



^Using seawater of salinity 25%oand pH 7.8 ± 0.1, 100 ± 4 

 eggs in 0.2 ml of seawater were added to 7.3 X 105 sperm in 

 0.07 ml of seawater; 5 repetitions/flooding time were used- 



'L - = Mean percent larvae losses 



(%Z - minus %0j^). 



watch glasses were stacked to reduce evaporation 

 and incubated at 27.0° ± 0.5°C. Because the number 

 of swimming lavae did not increase after 6 h 

 postfertilization time, the number of fertilized 

 eggs was obtained by counting unfertilized eggs 

 remaining on the bottom at 6 h and subtracting 

 this figure from 100 (the number of eggs originally 

 present). After 24 h we transferred the watch 

 glasses to a 4°C refrigerator; within 30 min the 

 D-shaped (straight-hinged) larvae settled to the 

 bottom and were easily counted. 



Although none of the 460 oysters examined 

 appeared hermaphroditic, sperm-free controls 

 were used in all experiments. We did not observe 

 fertilization in any of the controls. 



Results and Discussion 

 The relationship between K and the number of 



699 



