SEX AND BREEDING 49 



c. Sex Determination in Arbacia Punctulata 



Though there is no morphological difference in the sexes in Arbacia 

 there are several ways of distinguishing males and females. One method 

 is by forced extrusion of gametes from a gonopore (E. B. Harvey, 

 i94od). 



With a Luer syringe and very fine (no. 27) hypodermic needle, insert a drop of 

 sea water saturated with KCl, into one genital pore. The operation is most easily 

 done wfth an electric light shining from above on the aboral surface of the animal so 

 as to show up the genital pores, and under a binocular microscope. A fine glass 

 pipette, of slightly smaller bore than the genital pore can be used but is liable to 

 break. A few eggs or a little sperm will almost immediately begin to ooze out from 

 this pore alone, and the sex can be distinguished by the color (red $, white (^). The 

 shedding is stopped at once by placing the animal in a jar of still, not running, sea 

 water; leave for a few hours before returning to running sea water. The readiness 

 with which the Arbacia may be thus "sexed" depends somewhat on the maturity of 

 the animal and the time of year. 



In a second method a small amount of material is drawn out with a Luer 

 syringe (and no. 27 needle), or fine glass pipette, applied at the opening of one of the 

 genital ducts; it is examined in a drop of sea water under the microscope. As there 

 is a sharp bend in the gonoduct just beneath the surface (see Fig. 7 from Brown's 

 Invertebrate Types), care must be taken not to insert the needle into the coelomic 

 cavity instead of the gonoduct. Also the red color may be due not to eggs but to red 

 amoebocytes present in both sexes. 



The electrical method of determining sex (E. B. Harvey, 1952, 1953, 1954) is 

 by far the best for distinguishing male and female in Arbacia and other sea ur- 

 chins. An alternating current of 10 volts is passed through the animal, which will 

 at once shed its sperm or eggs. Ordinary 60-cycle alternating current can be used, 

 with the I lo-voltage, reduced to 10 volts by a transformer. Lead electrodes have been 

 found best, as they are non-toxic and are easily made from lead tubing or from heavy 

 lead wire. The electrodes are placed at any two points on the shell of the animal 

 which lies, aboral side up, covered with sea water. Almost immediately after the 

 current is passed, the eggs or sperm will exude from each of the five gonopores, the 

 sperm in a thin white thread, and the eggs in a thicker red (in Arbacia) thread, later 

 tending to clump (Plate XVI, Photographs 1,2). When the current is stopped, the 

 shedding ceases; but it begins again when the current is allowed to pass. In this way, 

 the sex of the animal can be quickly determined, and a few eggs or a little sperm 

 obtained without harming the animal; and the same animal can be used repeatedly. 

 The eggs, if removed at once, fertilize perfectly and develop normally. The method 

 is of great value in places where sea urchins have become scarce. 



The rapid response of the sea urchin is due to the presence in the walls of the 

 ovary and testis of a layer of smooth muscle cells which are stimulated by the electric 

 current, causing the walls to contract and force out the eggs or sperm. 



A much more elaborate electrical method was described several years 

 ago by Iwata (1950) for Mytilus and for a Japanese species of sea ur- 

 chin, Heliocidaris crassispina. The simple method described above has 

 also been used successfully on the sand dollar and on several species 

 of annelids. 



Sea urchins containing only immature eggs do not respond. In some 

 species a higher voltage may be required. 



