SUBSTITUTION OF AGENCIES FOR FERTILIZATION 447 



in its osmotic pressure. The increase of osmotic pressure required is 

 not great. If Sodium Chloride be employed an increase of forty per 

 cent, in the osmotic pressure of the sea- water suffices to initiate develop- 

 ment after an exposure of two hours. If sugar or urea be employed 

 even a slighter increase of osmotic pressure suffices to bring about a 

 like effect, because these substances penetrate the egg with greater 

 difficulty than the inorganic salts and hence exert a greater osmotic 

 tension on the external surface of the egg. The requisite concen- 

 tration of the medium varies, however, with the duration of the expos- 

 ure, a weaker concentration being effective after a longer exposure. 

 This is shown by the following experiment: To 50 c.c. portions of 

 artificial sea- water (Van t'Hoff's Solution) rendered favorably alkaline 

 by the addition of 2 c.c. of tenth normal sodium hydroxide were added 

 0, 2, 4, 8 and 16 c.c. of 2| normal potassium chloride solution. Unfertil- 

 ized eggs of a Pacific Ocean sea-urchin (Strongylocentrotus purpuratus) 

 were divided between these five solutions and samples removed after 

 varying periods of exposure and placed in normal sea-water. The 

 following were the results obtained: 



Increase in the osmotic pressure of the medium. 

 Period of exposure, 



minutes. per cent. 16 per cent. 30 per cent. 55 per cent. 87 per cent. 



45 .... No larvae No larvae No larvae No larvae Numerous 



larvae. 

 64 .... " " " Numerous 



larvae 

 89 . . .. .' " Numerous 



larvae 



116 ..'..;. 

 114 . . 



The fertilization which resulted from this procedure failed, however, 

 to furnish a perfectly faithful imitation of the phenomenon of natural 

 fertilization. It is true that the eggs frequently developed into free- 

 swimming larvae, but the larvae produced in this manner were sickly 

 and abnormal and did not survive very long. The percentage of eggs 

 which developed into larvae was variable and in some species, partic- 

 ularly in the sea-urchin Strongylocentrotus franciscanus, few if any of 

 the eggs could be induced to develop by this procedure. The larvae in 

 all cases behaved abnormally; they did not rise to the top of the water 

 and swim there as normal larvae do, but swam instead at the bottom of 

 the vessel containing them, and finally, the most marked peculiarity 

 of all was the failure of the eggs to form a Fertilization-membrane. 



If the eggs of a mature female sea-urchin be removed from the 

 ovaries by shaking them out in sea- water and are then mixed with 

 sperm similarly procured from the spermaries of a male, the sperma- 

 tozoa will immediately be seen clustering around the eggs, presenting 

 the appearance of striving to enter them. Within a very brief period, 

 under normal conditions, a spermatozoon will succeed in effecting an 

 entry, and this event is at once indicated by the appearance upon the 



