220 



Appendices to Fourth Annual Report 



containing the ova is orange-coloured. Many of the ova thus extruded 

 have a trauskicent investment, discernible on account of floating debris 

 being unable to reach the egg-capsule. Under the least pressure the ova 

 appear oval ; they are, however, quite circular when fully ripe, and at 

 that time the large germinal area and distinct germinal spot previously 

 seen are lost to view, the vitellus becoming densely granular. Viewed 

 by transmitted light under the microscope, they have a brown tinge; by 

 reflected light they appear as minute white specks, individually barely 

 visible to the naked eye. The pale genital canal on each side is readily 

 seen in the angle formed by the abdomen and the upper or attached 

 margin of the inner gill. Microscopic sections show it to be clothed in- 

 ternally by ciliated epithelium. Entering into the generative organ, it 

 divides into smaller canals which lead to the sperm- or egg-follicles. 

 The natural method of fertilisation is not yet fully inquired into. In all 

 probability the ova are carried by the efferent currents into the sur- 

 rounding water, and there fertilised by the spermatozoa similarly evacu- 

 ated. Artificial fertilisation is accomplished in the following way : — A 

 piece of tissue containing spermatozoa is minced in a watch-glass with sea- 

 water. A little of the milky liquid is poured into another glass and more 

 water added. Into this is poured some liquid containing ova procured in 

 the same way. The two elements are then mixed by gentle stirring, and 

 the glass is set in a cool place for half an hour. The milkiness must then 

 be completely removed by repeatedly emptying and refilling the vessel 

 with a pure supply of sea-water, allowing the ova to settle to the bottom 

 each time. Any debris left may be picked out by a fine pipette. With 

 patience this can be very successfully done, and speedy putrefaction will 

 be prevented. If the ova are now examined microscopically, each will be 

 found to have a considerable number of spermatozoa attached to it, and 

 their wriggling causes it to rotate. Fresh supplies of sea-water must be 

 given at intervals. In about four hours the first indication of successful 

 fertilisation is seen, the polar or direction-cell then making its appearance. 



The latest attempt to fertilise eggs was made on the 7th August, and it 

 failed. That of 1st August was partially successful. The most success- 

 ful effort was the first, made in the early part of June. The results as to 

 the time certain stages are reached in the different series are conflicting, 

 due no doubt to the decreasing vigour of the generative elements. It is 

 evident that as autumn advances the spermatozoa left in the organs lose 

 their activity, and the percentage of good ova is smaller. The first seg- 

 mentation takes place immediately after the appearance of the polar cell, 

 resulting in a larger segment (macromere) and a smaller (micromere). 

 Kepeated budding of the micromere takes place, and in six or seven hours 

 the embryo has assumed its most irregular condition, that is, it appears as 

 a large cell, round one pole of which are grouped many smaller cells. 

 Thereafter it assumes a form approaching the spherical, and in ten hours 

 the brownish granular contents give place to colourless translucent globules 

 and refracting particles. In fifteen or eighteen hours the shape is nearly 

 spherical, and the contour is broken by the projecting part of what in all 

 probability is the still undivided macromere. ' This body in some is almost 

 enclosed by the cap of cells which are presumably the product of micro- 

 meral segmentation. In others it may still project considerably, and give 

 indication of bipartition. The polar cell is usually now to be seen in the 

 periphery, asymetrically placed in relation to the macromere. An unex- 

 plained feature is the crenate outline of the projecting portion of the 

 latter. The gastrula stage is now reached, characterised by the appear- 

 ance of a large cavity lined by the micromeral spherules and many smaller 

 highly refracting cells. In gastrul^e of sixteen hours on more than one 



