274 MESSRS. C. SHEARER, W. DE MORGAN, AND H. M. FUCHS 



through bolting silk, or by means of a pipette. The spermatozoa were allowed to 

 float off the testes by placing a small portion of the latter in a bowl of " outside " 

 water. 



The rapidity with which the ova separate out of the ovarian tissue, when this is 

 gently shaken in a little sea-water, indicates, to a certain degree, the relative ripeness 

 of the eggs. The eggs when seen under the microscope should not show their 

 nuclei, and the cytoplasm should present a uniform homogeneous appearance, which, 

 although hard to describe, is soon recognised in practice. Once this appearance of 

 the cytoplasm of the ripe egg is learnt, it is perhaps the most satisfactory test of 

 relative ripe- or unripe-ness of the eggs. Spent or old ovarian tissue, towards the 

 end of the breeding season, dissolves into a mass of chalky substance when shaken 

 into water, and usually no eggs can be distinguished in this substance. The rapidity 

 with which the fertilization membrane is thrown out by the ova, when a little sperm 

 is added, is also an indication of ripeness, as the membrane should always appear 

 within a few minutes. 



The relative ripeness of a number of males is more difficult to determine than in 

 the case of the females. The ripeness of the sperm can be judged to a certain 

 extent by the amount which exudes from the cut surface of the testes. The only 

 real test, however, is that of comparing the relative motility of the spermatozoa in 

 a number of samples, and of choosing the most active of these. 



In making the fertilizations, it was found necessary to use an extremely small 

 quantity of sperm. Any excess of the latter speedily dies and putrefies in the water, 

 with the result that the fertilized eggs either die or produce unhealthy larvae. In 

 this work we have brought about all the cross-fertilizations without resorting to the 

 use of concentrated sperm, or of a changed chemical medium, as is sometimes 

 necessary when forms less closely related than those used in these experiments are 

 being crossed. 



It should be mentioned here, that all the observations recorded in this paper were 

 made on healthy material. Any cultures which showed a high percentage of 

 abnormalities in the early stages were rejected. In the late stages there are 

 necessarily, even in the best cultures, a few plutei which are stunted and malformed. 

 In making the observations these latter were neglected, since in such individuals 

 a given character may be poorly developed from other causes than those of heredity. 

 If such pathological individuals are taken into account, there must necessarily be 

 a confusion in the results. In a few cases, cultures showed a large proportion of 

 abnormalities in a late stage, and these cultures were discontinued. 



During the course of an experiment a freshly sterilized pipette was used for each 

 operation. After the experiment, all the glassware was thoroughly sterilized with 

 boiling water. 



Before a batch of eggs was fertilized, some were always removed to a separate 

 vessel as a control. During the whole course of the investigation, in only one or two 



