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

Abstract

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...

To paddle or not: context dependent courtship display by male blue crabs,Callinectes sapidus

“…This includes a courtship display by males towards a sexually receptive pubertal female crab (Ryan, 1966;Fielder and Eales, 1972;Berrill and Arsenault, 1982;Gleeson, 1991;Hardege et al, 2002). However, only the blue crab Callinectes sapidus (Teytaud, 1971;Gleeson, 1991;Wood and Derby, 1995;Jivoff and Hines, 1998) and the lesser blue crab Callinectes similis (M.K., personal observation) are known to have paddling as one component of the male display, in which they rhythmically wave their swimming legs.…”

“…Portunid crabs use sex pheromones and visual signals to communicate reproductive status (Ryan, 1966;Gleeson, 1991;Hardege et al, 2002). This includes a courtship display by males towards a sexually receptive pubertal female crab (Ryan, 1966;Fielder and Eales, 1972;Berrill and Arsenault, 1982;Gleeson, 1991;Hardege et al, 2002).…”

Identification of a female sex pheromone in Carcinus maenas

“…Guarding behavior continues through the molt until the female's carapace has hardened enough to provide protection and to discourage competitive mating, although guarding does not necessarily result in single paternity, as takeovers have been well documented in other crustaceans (for review see Jormalainen 1998). The same pre-copula males are not attracted to inter-molt females, and exposure to other males or inter-molt females evokes fighting behavior (Hardege et al 2002). This well characterized behavior is easily recognized, being very different from feeding responses (Hayden et al 2007).…”

“…Although Ryan (1966) demonstrated in an elegant series of experiments conducted more than 40 yr ago that female sex pheromones influence reproductive behavior in malacostracan crabs, the chemical nature of these cues remains unknown (Hardege et al 2002). This has been attributed to 2 problems: the biological assays are not able to effectively discriminate between signals derived from the opposite sex and other cues such as food (Dunham 1978(Dunham , 1988, and the analytical methods to purify pheromones from an aqueous environment are challenging (Hardege et al 2002, Hayden et al 2007).…”

“…This has been attributed to 2 problems: the biological assays are not able to effectively discriminate between signals derived from the opposite sex and other cues such as food (Dunham 1978(Dunham , 1988, and the analytical methods to purify pheromones from an aqueous environment are challenging (Hardege et al 2002, Hayden et al 2007). Many studies have described the existence of aquatic chemical cues as mechanisms that control animal behavior, but few aquatic sex pheromones have actually been identified.…”