SUBSTITUTION OF AGENCIES FOR FERTILIZATION 449 



does not occur until after the restoration of the egg to the normal 

 sea-water. 



The eggs which have been treated in this manner may undergo a few 

 divisions but they very rapidly die, more rapidly, in fact, than unfertil- 

 ized eggs exposed to similar conditions of temperature, etc. The 

 processes thus initiated therefore, still afford an incomplete analogy 

 to natural fertilization. It was found, however, that by a combination 

 of these two processes, membrane-formation and osmotic treatment, 

 which are separately incomplete, a perfect imitation of fertilization is 

 procured and a high percentage of the eggs, usually 100 per cent., can 

 be induced to develop and produce normal larvae. 



The precise details of time, of exposure, concentration and so forth, 

 in Loeb's improved method of Artificial Parthenogenesis necessarily 

 vary slightly with the temperature, reaction of the sea-water and 

 species of Echinoderm employed. The following are, however, the 

 details of the method as utilized for the fertilization of the Pacific 

 sea-urchin, Strongylocentrotus pur pur aim at a temperature in the 

 neighborhood of 15 C. The eggs, after extraction from the ovaries 

 and rinsing in filtered 1 sea-water are immersed in a mixture of 50 c.c. 

 of sea-water and 2.8 c.c. of tenth normal Butyric Acid solution, and 

 the mixture is gently agitated to prevent the eggs, which become sticky, 

 from adhering to the bottom of the vessel. After about two minutes the 

 eggs are collected by gentle rotation of the shallow flat-bottomed 

 vessel and transferred by means of a pipette to normal sea-water. If 

 the exposure has been rightly chosen it will be found that the eggs 

 almost immediately form membranes. After allowing them to remain 

 for some fifteen to twenty minutes in the normal sea-water they are 

 again collected in the manner described and transferred to Hypertonic 

 Sea-water, prepared by adding 8 c.c. of 2J molecular sodium chloride 

 solution to 50 c.c. of sea- water. They are exposed to this addition for 

 a period varying from fifteen to sixty minutes, the optimal exposure 

 varying somewhat with the eggs from different females. The eggs are 

 now returned to normal sea-water. Within about one hour the first 

 cell-division will be observed to have occurred, at the end of forty- 

 eight hours swimming gastrulse will have been produced, and about 

 two days later plutei with well-developed skeletons. 



Artificial fertilization has been extended to a variety of forms other 

 than the Echinoderms. In a number of Annelids development may 

 be induced by preliminary treatment with a cytolytic agent followed 

 by treatment with hypertonic sea-water. As a rule, however, in this 

 group the fatty acids do not constitute sufficiently potent cytolytic 

 agents, and saponins or, better still, mammalian blood serum must be 

 employed. Among the Molluscs simple treatment with hypertonic 

 sea- water frequently suffices, especially if it be rendered slightly hyper- 



1 The sea- water must be filtered to remove spermatozoa which may possibly be 

 suspended in it. 

 29 



