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. KEY WORDS: Sex pheromone · Shore crab · Bioassay · Carcinus maenas · 20-hydroxyecdysone Resale or republication not permitted without written consent of the publisherMar Ecol Prog Ser 244: [179][180][181][182][183][184][185][186][187][188][189] 2002 The existence of a female sex pheromone in crustaceans was first demonstrated in Portunus sanguinolentus (Ryan 1966). Since then, the list of crustacean species for which there is evidence of a sex pheromone has become extensive and still continues to grow (Dunham 1988, Bouchard et al. 1996. It includes the lobster Homarus americanus, where females orientate towards male burrows in response to sex attractants emitted by males and female-produced sex pheromones control mating behaviour (Cowan & Atema 1990). In the blue crab Callinectes sapidus, Gleeson (1980Gleeson ( , 1982Gleeson ( , 1991 and colleagues (Gleeson et al. 1984) described the presence of 3 pheromones in female urine that were attractive to males. Although some progress has been made in the purification of the latter (Gleeson et al. 1984), no sex pheromone has been characterised.The female sex pheromone is one of only a few crustacean pheromones so far demonstrat...

Volatile organic substances (VOS) were extracted from the coelomic fluid of gravid specimens of the lugworm Arenicola marina through the use of using a closed loop stripping technique and analysed via coupled gas chromatography-mass spectroscopy. VOS detectable in coelomic fluid of mature specimens include aldehydes, n-alkanes, methyl-alkanes, terpenes and ketones. The bouquet of compounds is very similar to nereid volatiles that have been recently described as having sex pheromonal activity. One of the major constituents is the ketone 5-methyl-3-heptanone, the sex pheromone of Platynereis dumerilii and Nereis succinea. Behavioural bioassays with gravld specimens produced no evidence that this substance has any pheromonal function in A. marina. Samples taken at monthly intervals showed an increase in the content of VOS in the coelomic fluid of both sexes during maturation with 1 major peak, 2-ethyl-hexanol, representing up to 50% of the total amount of VOS in spawning male lugworms. Behavioural assays with ripe females established the existence of a chemical cue inducing the burrow ~rngation behaviour during which sea water is pumped through the burrows. This behaviour ensures transport of spermatozoa from the water column into the tube inhabited by the female and increases fertilization success by minimising the dilution effects of the incoming tide. The VOS extract obtained from male spawning water had biological activity in eliciting the 'pumping' behaviour in females, with 16 of 24 worms irrigating continuously upon exposure, whilst controls showed no increase of activity, with 'pumping' occurring at 30 to 40 min intervals.