KITTREDGE. TERRY, and TAKAIIASHE ACTIVITY OF CRUSTECDYSONE 



near piadient of ethanol in water for the frac- 

 tionation of the phytoecdysones. We have found 

 that a column of XAD-2 could be used to re- 

 cover traces of crustecdysone from seawater and 

 from crab urine. Using the above two columns 

 we have examined the seawater in which female 

 P. crassipes, C. magistev. and C. productns had 

 been maintained for 3 to 6 hr. The product 

 of individual Cancer were assayed, while the 

 seawater from two or more P. crassipes was 

 combined before extraction. We have also ex- 

 amined the urine of female C. magister. The 

 Cancer were staged according to Drach (1939), 

 and the female P. crassipes were selected for 

 activity by observing the behavior of males in 

 their presence. During our observations we 

 found the female C. magister continued to re- 

 lease a pheromone for up to 2 weeks post molt. 

 The material recovered from the isolation 

 column in 60 ';r ethanol was reduced in volume 

 to a few microliters and transferred in 20';'r 

 ethanol to the Chromosorb 102 column. Elu- 

 tion of this column yielded an ultraviolet-ab- 

 sorbing peak near the front and two or more 

 succeeding peaks. Each of three stage "D" C. 

 magister and one stage "D" C. productns studied 

 were found to have released an ultraviolet-ab- 

 sorbing compound that eluted from the column 

 at the same ethanol concentration that a crus- 

 tecdysone standard did. Both extracts of P. 

 crassipes seawater yielded a peak in the position 

 of crustecdysone. One stage "A" C. magister 

 also yielded a peak in this position. A stage 

 "C-4" C. magister did not yield any peak in 

 the position of crustecdysone nor did urine from 

 a stage "C-4" C. magister yield a peak in this 

 position. Also one of the C. magister females 

 that had yielded material eluting as crustecdy- 

 sone while in stage "D" did not yield this sub- 

 stance during molting (stage "E"). The ultra- 

 violet-absorbing fractions corresponding to 

 crustecdysone from the above columns did not 

 have an ultraviolet-absoi'ption spectrum corres- 

 ponding to that of crustecdysone. The absorp- 

 tion peak included the spectrum of crustecdy- 

 sone, but had a double peak at a lower wave 

 length. These fractions were concentrated and 

 applied to a silicic acid column. The elution of 



this column with chloroform-ethanol yielded an 

 ultraviolet-absorbing peak near the front and 

 a peak eluting in the same volume as a crus- 

 tecdysone standard. The material from this 

 column has an ultraviolet-absorption spectrum 

 that corresponds closely to that of crustecdysone. 

 We are now developing a derivitization tech- 

 nique that will permit us to subject our final 

 samples to gas chromatography-mass spectro- 

 photometry for structural confor'mation. Katz 

 and Lensky (1970) have published a technique 

 for the silylation of a-ecdysone for GLC analy- 

 sis. We have employed silylation techniques 

 with crustecdysone and observed decomposition 

 during GLC. 



RESULTS AND CONCLUSIONS 



The pheromone released by P. crassipes stim- 

 ulates premating behavior in C. antennarius. 

 Male C. magister are excited into seizing and 

 clasping female C. productns by their phero- 

 mone. Crustecdysone mimics the pheromone in 

 its effects on male P. crassipes, C. antennarius, 

 and C. anthonyi in the release of the premating 

 stance. After exposure to crustecdysone all 

 these species of male crabs attempt to seize 

 other crabs, male or female, and pull them into 

 a precopulatory position. In addition crustec- 

 dysone triggers a search behavior in male C. 

 antennarius. These observations demonstrate 

 a lack of specificity in the sex pheromones of 

 these species and either that crustecdysone is 

 the sex pheromone or sufficiently similar in mo- 

 lecular structure to the natural pheromones to 

 mimic them. 



A possible explanation of the discrepency be- 

 tween our results and those of Ryan (1966) 

 that indicated a species specificity for the sex 

 pheromones in the three species of crabs that 

 he studied may be that some species may re- 

 spond to deoxycrustecdysone, callinecdysone A 

 (inokosterone) or callinecdysone B (makister- 

 one), other ecdysones that have been isolated 

 from Crustacea, (Gailbraith et al, 1968; Faux 

 et al., 1969), or they may respond to one of the 

 metabolic products of crustecdysone detected in 

 insects (Gailbraith et al, 1969; Moriyama et al., 



341 



