KITTREDGE, TERRY, and TAKAllASHI; ACTIVITY OF CRUSTECDYSONE 



This aspect of their behavior could be inter- 

 preted as a courting gesture, but in the context 

 of chemical communication we prefer to inter- 

 pret this as a fanning motion facilitating the 

 distribution of pheromones that may be aphro- 

 disiac in nature. Commercial fishermen for 

 both the American lobster, Homarus american- 

 its. and the California spiny lobster, PanuUrus 

 interrtiptus, have suspected that the males of 

 each species could be used to attract the females. 



A single active premolt female Parhygrapsus 

 crassipes released sufficient pheromone to stim- 

 ulate all of the males in a 25-gal recirculating 

 aquarium. A single female P. crassipes in an 

 aerated 2-gal container released a phei-omone 

 which stimulated a male Cancer antennarins 

 to exhibit a premating stance. 



With these observations providing the bio- 

 assay, we examined the nature of the sex pher- 

 omone. Water which had contained a premolt 

 female crab was boiled for 10 min, cooled, and 

 aerated. The active principle was still present. 

 The active principle was not retained by cation 

 nor anion exchange resins nor by charcoal. The 

 active substance could, however, be extracted 

 from "active seawater" with isopropanol/di- 

 ethyl ether. These observations, together with 

 the premolt condition of the active females, led 

 us to suspect that the females might be releasing 

 molting hormone into the water, and that this 

 steroid might be functioning as a sex pheromone. 



BIOASSAY OF CRUSTECDYSONE 



We soon confirmed that dilute solutions of 

 crustecdysone (/3-ecdysone, 20R-hydroxyecdy- 

 sone, ecdysterone, isoinokosterone) , which is 

 one of the molting hormones of Crustacea (Horn 

 et al., 1968), elicited a typical response from 

 male P. crassipes. In order to establish the 

 threshold concentration that would release re- 

 sponse we standardized the conditions for the 

 bioassay. As described under "Methods," the 

 test adopted allowed the male crab to be flooded 

 with a known concentration of the steroid in 

 seawater, in contrast to the diffusion techniques 

 often em]3loyed. P. crassipes proves to be ideal 

 for this mode of testing because they are an 



intertidal species and in nature normally leave 

 the tide pools at low tide to feed on the rocks. 

 They were not disturbed on being placed in a 

 wet empty observation vessel and sought out 

 the artificial niche provided. On flooding with 

 seawater they would remain in the niche for 

 long periods or occasionally come out to explore 

 briefly and then return to the niche. When a 

 male P. crassipes was flooded with a solution 

 of crustecdysone in seawater, he was stimulated 

 to come out of the niche and explore the vessel 

 and then to assume the premating stance. The 

 time elapsing until the crab raised its body to 

 assume the stance was found to be a function 

 of the concentration of crustecd.vsone. Although 

 the male crabs would often exhibit a full stance 

 in a brightly illuminated laboratory at the high- 

 er concentrations of crustecdysone, the response 

 was often erratic. All of the bioassays were 

 conducted in an isolated room illuminated with 

 an Eastman darkroom lamp with a 1.5-w bulb 

 and an Eastman No. 00 yellow filter. The ob- 

 server was stationed quietly before the "win- 

 dow" of the test vessel. On removal from the 

 test vessel the male crabs were transferred to 

 a 2-liter beaker of seawater to rinse off the ex- 

 traneous crustecdysone and then were trans- 

 ferred to a small aquarium for further obser- 

 vation. In spite of the handling during the two 

 transfers, male crabs that had been stimulated 

 to display a premating stance in the observa- 

 tion vessel usually resumed this posture shortly 

 after being transferred to the aquarium. When 

 thus stimulated they often attemjited to seize 

 any other male crab in the aquarium. 



All of the male P. crassijies utilized in estab- 

 lishing the response curve were collected at the 

 same time and in the same area where we had 

 just succeeded in collecting a number of in-e- 

 molt females. They were all held for three or 

 more days in isolation from any female crabs. 

 The curve was started with a concentration of 

 10^'' M crustecdysone in filtered seawater. At 

 this concentration the resjionse was rapid, aver- 

 aging 7 sec. Succeeding tests were performed 

 with ten-fold dilutions of the crustecdysone. 

 Fresh solutions of crustecdysone were prepared 

 after three steps of dilution or at the start of 

 each day's testing. Previous experience had 



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