122 Papers from the Marine Biological Laboratory at Tortugas. 



The failure to make a thorough attempt at the determination of 

 the necessary conditions for fertilization by foreign sperm has been due, 

 in my own case, to the fact that it seemed of advantage to make immediate 

 use of the crosses that were readily made and to delay until some future 

 time the investigation of methods necessary for making other crosses. 



METHODS OF TREATING ECHINOID EGGS BEFORE FERTILIZATION. 



The methods for increasing the number of fertilized eggs in hybrid 

 cultures which have been most employed are : (i) Treatment with 

 alkalis. (2) Treatment with fresh water. (3) Subjection to increased 

 temperature. (4) Excess of sperm. (5) Allowing eggs to stand. 



The method of Loeb has been most used, i.e., treatment of the eggs 

 with an alkali before fertilization. This method has been used with 

 signal success by Herbst and Godlewski. 



Herbst (1906, p. 183) obtained his best results by adding between 

 one and two drops y"^ NaOH to 20 c.c. sea-water, and further pointed out 

 that the optimum concentration is an individual affair. 



Doncaster (1903) found that the percentage of fertilizations was 

 increased in diluted sea -water. Herbst (1906) also tried the effect of 

 fresh water, allowing the eggs to remain in fresh water i to 3 minutes. 

 Some of the eggs were destroyed, but most remained unharmed by the 

 treatment. As to the value of this method when compared with others 

 mentioned, he does not feel prepared to decide. 



Herbst's (1906a) experiments on the influence of warmth are of 

 interest. In general it seemed that the optimum was about 24° C. 



Born's method of excess of sperm is of somewhat doubtftil value 

 when used for Echinoderm crosses. With this method polyspermy is 

 the usual result. 



The method used by the Hertwigs (1885), of allowing the eggs to 

 stand for some time before fertilization, has been used by several inves- 

 tigators. Vernon was at first inclined to regard it as a most useful 

 method, but later came to look upon it as of somewhat doubtful value, 

 especially when one desired older larvae. Driesch also does not regard 

 the method favorably. In my own work it is the method that I have 

 employed almost exclusively and it has given me exceptionally good 

 resiilts. I have found that there is an individual or more properly a 

 species optimum for the length of time that the eggs should stand. 

 For example, with the eggs of Arbacia, Hipponoe, and Toxopneustes, 

 with which most of my work has been done, three different periods of 

 time were allowed to elapse before the eggs were fertilized. 



The Arbacia eggs were fertilized with Moira sperm 7 hours after 

 their removal from the ovary. The Hipponoe eggs were best fertilized 

 with Toxopneustes sperm 2^ hours after removal from the ovary. The 

 Toxopneustes eggs were best fertilized with Hipponoe sperm 6 hours and 

 with Moira sperm (at Beaufort) 5 hours after removal from the ovary. 



I have tried other methods which will be mentioned in other parts 

 of this paper, but never with the success that followed that of allowing 

 the eggs to stand. The percentage of fertilization following this treat- 

 ment in the Arbacia, Hipponoe, and Toxopneustes crosses was from 75 

 to 95 per cent. 



