Novel behavioural assay and partial purification of a female-derived sex pheromone in Carcinus maenas

Experiments were conducted to test the hypothesis that females of the shore crab Carcinus maenas release a sex pheromone to induce the pre-copula behaviour leading to the formation of mating pairs. In the novel behavioural assay, a positive response to a female-derived signal involves 'homosexual' behaviour: a pre-copula male moves towards another pre-copula male that has been 'exposed' to the substance(s) under investigation. The assay male grasps the 'pheromone-treated' male (the 'pseudo-female') to test the hardness of the cuticle with its chelipeds, and invariably proceeds to manipulate the pseudo-female beneath its abdomen as in pre-copula ('homosexual pair formation'). Control males show an agonistic behaviour towards each other. Male shore crabs will also attempt to pair with non-crab-like objects, such as stones, if these are conditioned with female pheromone. This demonstrates that male mate selection in Carcinus maenas is based primarily on the detection of female odour compounds. Urine collected from the urinary bladder of pre-copula females, as well as 'culture water' obtained from these females, elicited a positive response, whereas similar samples collected from inter-moult females failed to induce a sexual response in males. Preliminary characterisation of the 'waterborne cue(s)' revealed the sex pheromone(s) to be smaller than 1000 Da molecular weight, sparingly soluble in organic solvents, lyophilisable, and chemically distinct from 20-hydroxyecdysone (crustecdysone) and arthropodin. Crustecdysone showed no biological activity in the assay and was not detectable in the bioactive HPLC fraction. Although this does not completely rule out a steroidal character for the sex pheromone, LC-MS analyses utilising electrospray and atmospheric pressure chemical ionisation (APCI) did not show a steroid in the bioactive fraction.