Isolation and characterization of alarm pheromone from electric shock-induced earthworm secretion

Electric stimulation of earthworms, Lumbricus terrestris, causes secretion of a yellow mucus which has alarm properties for conspecifics and chemoattractive properties for garter snakes. An alarm pheromone was isolated and purified to homogeneity from the mucus by means of permeation, thin-layer and high performance liquid chromatographies. The purified substance was highly active as an alarm chemosignal to earthworms (L. terrestris), but it did not elicit alarm responses from either sandworms (Nereis virens) or bloodworms (Glycera debranciata). It was not a snake chemoattractant. The alarm pheromone could not be retained with 1 kDa cut-off dialysis tubing, and it was eluted from a Bio-gel P2 column ahead of p-nitrophenol. These data suggest an apparent mass greater than 139 Da but less than 1 kDa. The order of solubility of this alarm pheromone is H2O greater than DMSO greater than MeOH greater than 2-propanol greater than acetone. It was thermostable, and it fully retained activity after heating at 100 degrees C for 1 hour. This alarm pheromone fluoresced under u.v. light, and it showed an optimal excitation wavelength of 420 nm and emission wavelength of 465 nm.