96 E. E. JUST. 



Two hours after insemination the eggs exhibit cytoplasmic 

 stratification ; the oil drops later fuse to form one at the vegetative 

 pole. Twelve hours after insemination the conditions are the 

 same; there is never a swimming form among these eggs. 



2. Nature of the Inhibition to Development. 



It may be very clearly shown that sea-water is responsible for 

 the lack of cleavage by the method of "dry insemination." If 

 males and females dried on filter paper be cut up separately and 

 the drops of eggs and sperm thus obtained be mixed with sub- 

 sequent addition of sea-water, a percentage of the eggs always 

 cleave and develop into normal trochophores. I have kept larvae 

 from such dry inseminations until they were seven mm. long 

 with thirty or more segments, few differing from normally laid 

 eggs. There is doubtless an optimum time after mixing for the 

 addition of sea-water, but any time upward to two minutes 

 gives results. The following is an example: 



August 3, 1912. To determine the time interval after mixing 

 dry eggs and sperm before adding sea-water. 



Per Cent. 

 Water Added. of Cleavage. 



1. At once 60 



2. Five seconds after 50 



3. Ten seconds after 90 



4. Twenty seconds after 45 



Practically, as soon as eggs and sperm are mixed, sea-water 

 may be added. I have not been able to add sea-water quickly 

 enough after mixing to prohibit cleavage. If the eggs are allowed 

 to stand two minutes the majority are plasmolyzed by the addi- 

 tion of sea-water. 



The amount of sea-water that will permit fertilization has been 

 repeatedly determined: 



July 28, 1912, 9:45 P.M. Experiment to determine the maxi- 

 mum amount of sea- water that permits fertilization. 



Males and females are thoroughly dried on clean filter paper. 

 A male and a female placed in each of the eight perfectly dried 

 clean watch glasses. Sea-water added as follows: 



