Approaches to a Molecular Identification of Sex Pheromones in Blue Crabs

Abstract

Molecular identification of sex pheromones in marine crustaceans has proven to be very difficult, and so far no unequivocal identification for any decapod crustacean has been published. Some of these difficulties are common to other animals – pheromones are often blends of molecules at low concentrations. Some difficulties are more specific to marine crustaceans – pheromones are often small and polar molecules that are difficult to separate from salts in their source (often urine) or carrier medium (sea water). These difficulties led us to take on new approaches as we searched for sex pheromones in the blue crab Callinectes sapidus. Premolt pubertal female blue crabs that are ready to mate release a pheromone in their urine. This pheromone is detected by male crabs using specific chemical sensors – aesthetasc sensilla on the antennules. Male blue crabs respond to the pheromone with courtship stationary paddling, a distinctive behavior that is useful in bioassays for pheromone identification. We used bioassay-guided fractionation to demonstrate that the pheromone of female blue crabs is of low molecular mass (<1,000 Da) and possibly a mixture. We used liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance, biomarker targeting, and metabolomics approaches to isolate molecules specific to premolt pubertal females and that are thus candidate pheromones. Our working hypothesis is that female blue crabs release a species-specific sex pheromone in their urine that is composed of two functional classes of molecules, both of which are small and polar. One class distinguishes females from males and thus is a sex-specific signal, and a second class distinguishes blue crabs from other species and thus constitutes a species-specific signal.