ON THE EXPEEIMENTAL HYBEIDIZATION OF ECHINOIDS. 271 



The breeding periods of the three species are as follows : — • 



E. escidentu^ breeds from the end of February to June 



A', ncutus breeds from May to August. 



E. miliaris breeds from April to September. 



Finally, during our work we have often noticed the peculiar condition of our egg- 

 material that may be called ovt'y-ripeneas. In this condition the ova are to all appear- 

 ances perfectly ripe — yet they will not fertilize even with sperm of their own species. 

 It woidd seem that over-retention of the eggs within the ovary brings about some 

 change in the egg membrane which renders subsequent fertilization impossible. These 

 eggs show slight if any fertilization membranes on fertilization, and segmentation, 

 if it takes place, seems to be independent of membrane formation. In many 

 instances eggs in this condition will fertilize with foreign sperm when they will not 

 fertilize with that of their own species. In the vast majority of cases, however, the 

 eggs will not form membranes or fertilize at all, and there is nothing more trying 

 to the patience of the experimenter in Echinoderra hybridization work than the 

 misfortune of obtaining successive batches of material in this condition. 



4. Physical Conditions of Sea- Water, etc. 



In the following section we give a description of the sea- waters we have used in 

 the course of our experiments. We shall designate these for the sake of convenience 

 as " outside," " Berkefeld," and " tank " water. 



(1) In using the term " Outside " water we always mean sea-water collected in 

 large glass carboys of from 3 to 4 gallons capacity, obtained outside the more land- 

 locked portion of Plymouth Sound, in the region of the open (Jhannel, some five to 

 seven miles from land, and brought into the laboratory. This water is obtained 

 at the surface, and sometimes contains floating fragments of Fucus and other marine 

 algse ; for this reason it was sometimes filtered through several layers of fine filter- 

 paper before use. In it, in the unfiltered state, we liave never observed the presence 

 of plutei. What we had more particularly to guard against in the use of this 

 water was its possible contamination with sperm. By allowing the water to stand 

 for some time in the laboratory before use we were able to avoid this, as we found 

 fi'om experiment that the sperm of Echinus do not remain alive in it for more than 

 24 hours. Thus there need be no fear of containination from foreign sperm if the 

 water has stood at least three or four days in the laboratory before being used. All 

 our " outside" water was allowed to stand at least a week before being used, and 

 in the majority of instances two weeks and sometimes three. We have never had 

 any of our controls of unfertilized eggs go wrong from the use of this water, so 

 we feel certain no contamination could arise from tliis source. To make doubly 

 certain, however, during the last two years of our work we have taken the extra 

 precaution of filtering all " outside " water through a Berkefeld filter. The only 



