An electrophysiological and chemical investigation of the female sex pheromone of the crab Portunus sanguinolentus (Herbst)

Abstract

Chemoreceptors sensitive to the sex pheromone of Portunus sanguinolentus (Herbst) were examined using behavioral, histological and electrophysiological techniques e Certain physical and chemical characteristics of the molecule were also defined. The inner flagellum of the antennulae of male P. sanguinolentus was found to be sensitive to the sex pheromone of the premolt female. This was indicated by ablation experiments where males without their inner flagella failed to respond to the female pheromone with the characteristic display (which was the bioassay) described by Ryan (1966). Mature males did not give the display while a premolt female was being carried by another male or artificially held. This indicates that the female had some mechanism for controlling the release of the pheromone. In these cases the pheromone was still present in the urine of the female. It was established that while she was being carried the female inhibited emission of the sex pheromone by regulating her urine flow. Histological studies on the inner flagellum of the antennulae, using both silver, and haematoxylin and eosin methods, revealed that the hairs are innervated by numerous dendrites from groups of bipolar cells. The innervation greatly resembles other known crustacean and insect chemoreceptors. Electrical activity recorded externally from the inner flagellum of the antennulae demonstrated that the chemoreceptors respond to a wide array of pure chemicals. Flagellar receptors of mature males also appear to be able to differentiate urine with and without the female sex pheromone by a change in electrical activity. Female flagellar receptors appear unable to make such a distinction. The sex pheromone was stable, remaining stimulative after 15 minutes at 100°C. and 2 months at -20°C. The pheromone was soluble in absolute ethanol, but not in N-butanol, ethyl acetate, diethyl ether or petroleum ether. Size of the molecule was estimated to he close to 1000 molecular weight using dialysis, pressure ultrafiltration, am exclusion properties of G-15 Sephadex. The pheromone was separated with one-dimensional and two-dimensional ITLC-sa electrophoresis. Migration was towards the cathode in solutions ranging from pH 2.00 to 10.85. The presence of the sex pheromone was demonstrated by eluting from the electropherogram with Pantin's crab saline and testing the elutant on test males. There was a decrease in mobility between pH 4.00 and 7.25. The active fraction was eluted from the cation exchanger, SE-Sephadex at pH 6.90. The decrease in mobility and elution at pH 6.90 suggests that the isoelectric point lies between pH 4.00 = 7.25. The use of selected chemical detecting methods on electropherograms failed to give any conclusive chemical identification of the sex pheromone.