ON THE EXPERIMENTAL HYBRIDIZATION OF ECHINOIDS. 273 



the trouble experienced from overgrowth of micro-organisms as when " outside " 

 water is adopted. Its alkahnity is low, being on the average about Pn = 7 '40. 

 We have noticed that it is difficult to get a high percentage of fertilizations in this 

 water if it is used exclusively when effecting the fertilization. 



The rate of segmentation in it seems to be slower than in " outside " water. It 

 is certainly more difficult, as one of our number has observed [Shearer and 

 Lloyd (81)], to bring about artificial parthenogenesis in the Echinoderm egg in 

 this water. This is probably due to its low alkalinity. We have always used 

 "outside" water, therefore, for making the fertilizations; when the eggs have 

 reached the blastula stage, they have been pipetted ofi" into " Berkefeld " water, and 

 kept there till after metamorphosis, when they have been transferred once more to 

 " outside " water. 



(3) By " tank " water we meaji sea-water taken from the supply circulating 

 through the tank of the Aquarium at Plymouth. We have only made use of this 

 water for preparing our " Berkefeld " water and our dishes of food for the young 

 urchins, by means of Miquel's solution, as described in the following section. The 

 character of this water has been fully considered by Allen and Nelson (3), so we 

 need not describe it at length. Its alkalinity is low, being about Pn = 74; it is, 

 however, subject to considerable variation. Its salinity is comparatively constant, 

 being about S = 34 '9 per cent. 



5. Methods. 



Throughout the course of the experiments the same general methods have been 

 followed, and rigorous care has always been taken to avoid possible contamination 

 with foreign sperm. Material was obtained fresh each day, being brought in by the 

 steamer belonging to the laboratory. In 1912 we had to depend mostly for our 

 supply of E. esculent'us and E. acutus on the Plymouth trawlers, as the laboratory 

 steamer was not running. Our material of E. miliaris was obtained by shoi-e 

 collecting during low tides. We took precautions to have all our material as fresh 

 as possible, and the urchins were opened soon after being brought into the laboratory, 

 and usually within a few hours after being collected. 



Before being cut open, each Echinus was immersed in running fresh water to 

 destroy any sperm which might be adhering to the outside of the test. The scissors 

 were thoroughly sterilised between each operation. After the urchin had been 

 opened, the ovaries or testes, as the case might be, were removed by means of 

 a flat watch-glass, care being taken to avoid all contact with metal. The gonads 

 were placed in glass finger-bowls containing "outside" water. The gonads from 

 each individual were, of course, kept distinct, the sperm from a single individual 

 being used to fertilize the eggs from another single individual in each experiment. 

 The eggs were separated out from the ovaries by gently shaking the latter in the 

 water. The pieces of ovary were then removed either by filtration of the eggs 



VOL. CCIV. — B. 2 N 



