FISHERV BULLETIN, \0L. 69, NO. 2 



demonstrated that dilute solutions in seawater 

 lost some or all of their activity on storage even 

 at 0° C, presumably through bacterial degra- 

 dation or adsorption. It was planned that six 

 male crabs would l)e tested at each concentration 

 and that the five most consistent times would 

 be averaged; however, the response was found 

 to be remarkably uniform and in all but three 

 cases all six crabs responded within a narrow 

 time range. There was no sharp threshold of 

 concentration. The average response times 

 jilotted as a smooth curve extending to lO"''^ M 

 crustecdysone concentration where the average 

 response time was 22 min (Figure 1). No re- 



CRUSTECDYSONE 



500 1000 1500 



REACTION TIME (sec) 



Figure 1. — Time elapsed following immersion of male 

 Paehygrapsus ci'assipes in seawater solutions of crus- 

 tecdysone before the body elevation phase of the pre- 

 copulatory behavior. 



sponse was observed at 10"'" M. The scatter 

 of response times was greatest at 10"^ M, prob- 

 ably because of the short-term di.sturbance of 

 the male crab during flooding. The standard 

 deviation of the normalized response times for 

 '15 crabs, from 10 " m to 10 '■' M crustecdysone, 

 was 8.6. 



Since the ijrecopulatory l)ehavior of males in 

 the presence of premolt females ajipears to 1)C 

 general among the Brachyui-a, we examined the 



response of two species of Cancer to crustecdy- 

 sone. Both C. antennaruis and C anthonyi dis- 

 played typical premating behavior when exposed 

 to dilute solutions of crustecdysone. When the 

 respon.^e time vs. concentration study was car- 

 ried out with these two species each yielded a 

 response curve similar to that developed by P. 

 cmssipes. There was, however, a marked dif- 

 ference. There was an abrupt break in the re- 

 sponse yielding a distinct threshold at lO"'" M 

 for C. antenna I i us and 10~* M for C. anthonyi. 

 We then attempted to determine if C. anten- 

 na rius males could detect a gradient in the con- 

 centration of crustecdysone. For this i)urpose 

 we employed a simple "T" maze with a baflle 

 at the head of each arm separating the seawater 

 sources and forming mixing chambers. Five 

 male C. antennarhis were placed at the head of 

 the maze and the water flows from each arm 

 balanced. WHiile seawater alone was flowing 

 through the maze the C. ontennarhw remained 

 quiet in the two corners at the origin of the 

 maze. When a flow of crustecdysone solution 

 was added to the mixing chamber at the head 

 of one arm all five of the male C. antennarhis 

 soon became active. They ex])lored up and down 

 each arm of the maze but demonstrated no 

 tendency to select the arm containing the crus- 

 tecdysone. While these tests did not indicate 

 any ability to detect or follow up a gradient, 

 there was a positive re?i3onse to the crustecdy- 

 sone. All five of the male crabs were stimu- 

 lated simultaneously to undertake an active ex- 

 ploratory behavior when the crustecdysone was 

 introduced. 



DEVELOPMENT OF THE TECHNIQUES 

 FOR THE ISOLATION AND FRAC- 

 TIONATION OF THE SEX 

 PHEROMONE{S) 



Liquid-liquid extraction procedures are inef- 

 ficient for the recovery of trace quantities of 

 polar lipids. Columns of Amberlite XAD-2 have 

 been employed for the recovery of steroids from 

 urine (Bradlow, 1968; Shackleton, Sjovall, and 

 Wisen, 1970). Recently, Hori (1969) has em- 

 ployed a column of this resin eluted with a li- 



340 



