STUDIES OF FERTILIZATION 547 



tions at first showed radiating strands of sperm visible to the naked 

 eye, but then closed to form a solid drop, and this grew at its 

 margins, preserving the clear external zone, until the clear margins 

 of the two drops, at first several miUimeters apart, ran together. 

 Outside of the influence of the drops cloud formations appeared, 

 corresponding to an early stage of the aggregations of Nereis. 



The clear margin of the drops is not by any means so well 

 defined as in Nereis. Moreover, the spermatozoa are so small 

 that it is difficult to observe their behavior in the clear margin. 

 However, there can be no doubt that the phenomenon is essen- 

 tially the same as in Nereis, and that the aggregation in the drop, 

 the appearance of the clear margin, and the growth of the aggrega- 

 tion are due to positive chemotaxis to COo. 



This reaction is given clearly only by a fresh sperm suspension. 

 One ten minutes old does not give it, owing presumably to for- 

 mation of CO2 in the suspension. 



A considerable number of tests were made. In some the reac- 

 tion was much more rapid than in the experiment described. 

 In one of these tests I injected drops of 20 per cent, 4 per cent and 

 1 per cent of the CO2 sea-water near together. In the case of the 

 20 per cent a ring with external clear margin was formed in a few 

 seconds. The ring did not close. The 4 per cent formed a ring 

 which closed in its center. There was no reaction to the 1 per 

 cent. The same suspension gave no reaction twenty-five minutes 

 after it was mixed. In another case I got a faint reaction to 

 1 per cent CO2 sea-water. 



The fact of positive chemotaxis of Arbacia sperm to CO2 

 dissolved in the sea-water was repeatedly demonstrated. In 

 the case of a drop mounted beneath a raised cover it expresses 

 itself by a gradual aggregation of the sperm towards the center, 

 leaving the margins clear. 



As is to be expected from the slower and less delicate reaction 

 to CO2, as compared with Nereis, spermatozoa of Arbacia react 

 also to other acids, but more slowly and not to so great dilutions. 

 Thus in tests of N/10, N/50, N/250 and N/1000. H.SO,, strong 

 positive reactions were obtained for the first three, whereas only 

 a faint shadowy reaction is given to N/1000. In the case of Nereis 



THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 14, NO. 4 



