STUDIES OF FERTILIZATION 539 



spermatozoa exhibit the aggregation reaction in any marked 

 form so far as I know; Arbacia which comes next to Nereis in 

 point of sensitiveness to CO2 among the forms studied, shows, 

 under certain conditions, a cloud-like formation similar to the 

 initial stage of aggregation in Nereis. These will be referred to 

 beyond. 



It may perhaps be objected that the aggregation reaction in 

 Nereis is not necessarily caused by CO2 excretion, but possibly by 

 some other substance produced by the spermatozoa. And it 

 would be difficult to meet this objection in any absolutely con- 

 clusive way. But the following considerations render the con- 

 clusion extremely probable. In the first place it can be proved 

 that the spermatozoa exliibit positive chemotaxis towards some 

 substance that they themselves produce. Thus July 31, 1912, 

 the following experiment was made: 



With the dry sperm of one individual two suspensions were made 

 at the same time (8.57 a.m.) namely: a one drop of sperm in 9 cc. sea- 

 water, b two drops of sperm in 6 cc. sea-water; the activity of the sperm 

 in both suspensions being the same b should produce any attractive sub- 

 stance in much greater amount than a. This was tested by making 

 preparations of a and b on separate slides beneath raised covers. A drop 

 of b was then injected into slide a, and a drop of a into slide b at 8.59. 

 On slide a there was a very quick beautiful positive reaction to the intro- 

 duced drop, that is a clear border formed about the drop owing to positive 

 chemotaxis of tte spermatozoa a to the drop of denser suspension b. 

 On slide b not only was such positive reaction absent, but the drop of 

 introduced sperm actually lost its spermatozoa and became clearer, 

 owing to the positive chemotaxis now being away from the drop. The 

 same results were obtained also by using two suspensions of equal den- 

 sity, one of which was older than the other. The fresher suspension 

 reacted positively to the older suspension. 



In the second place, it is of course certain that the spermatozoa 

 becoming suddenly active in the sea-water must produce COo; 

 and as we have seen that the spermatozoa of Nereis react even to 

 a 1/200 dilution of a saturated solution of CO 2 in sea-water, if it 

 can be proved that a standard suspension of spermatozoa produces 

 an equivalent amount, the probability that CO2 is the agent 

 involved in the aggregation effect become very great. Experi- 

 ments directed to this end showed that a 1/100 dilution of CO2 



