540 FRANK R. LILLIE 



lies near the limit of demonstrability both by color reaction tests 

 and also by gas-burette estimation. A number of tests of sperm 

 suspensions were made with acid color indicators. In the case of 

 neutral red a dilute solution in sea-water has a decided orange 

 tinge due to slight normal alkalinity of the sea-water. The same 

 dilution made from a standard concentrated solution by the addi- 

 tion of a sperm suspension, shows a decided rose color without any 

 trace of orange. The spermatozoa then aggregated in the vial 

 used and the aggregations sank to the bottom, forming a bright 

 red precipitate, and the supernatant fluid, now merely opalescent 

 on account of the few sperms remaining in it, was faint rose. 

 There is thus a decided acid reaction of the sperm ^spension. 

 Tests with azolitmin and tropaeolin 000 No. 1 also gave clear indi- 

 cations of acid. The sperm suspensions were tested within two 

 minutes or less after their preparation; the liberation of the acid 

 takes place therefore very suddenly. It is liberated only when 

 the sperm become active, and the change of color is not given if 

 the sperm remain inactive. It is therefore very probable that CO2 

 is the acid revealed. 



Finally a large number of tests for CO 2 were made of the air in 

 closed flasks containing considerable quantities of active sperm 

 suspensions of Arbacia. The details of these tests made with a 

 gas burette need not be given. They extended over a week, using 

 the sperm of Arbacia which could be obtained in larger quantities 

 than Nereis. Although the determinations came very near the 

 limits of experimental error, there could be no question as to the 

 presence of CO 2 in quantities above that contained in the air 

 or in normal sea-water. 



In consideration of the facts (1) that sperm suspensions of 

 Nereis produce a substance to which spermatozoa of Nereis 

 react positively (2) that an acid is present in the suspensions (3) 

 that the production of CO2 by the suspensions can be demon- 

 strated and (4) that spermatozoa of Nereis react positively to 

 dilutions of CO 2 in sea- water which are barely detectable by 

 color indicator, or gas burette, it can hardly be questioned that 

 the aggregation reaction in Nereis is due to positive chemotaxis 



to CO2. 



