Earthworms (Lumbricus terrestris) produce a chemical substance that is readily detected by and serves as an attractant for garter snakes (Thamnophis sirtalis). This chemoattractant is sensed by the vomeronasal system ofsnakes. Amino acid analysis of the chemoattractant revealed a high hydroxyproline/proline ratio and large amounts of serine and threonine. More than one-third of the residues were glycine. No hydroxylysine and no cysteine were present. Carbohydrate analyses revealed a high content of galactose (11% by weight) and smaller amounts of fucose, mannose, glucose, N-acetylglucosamine, and N-acetylgalactosamine. These results were very similar to results reported for the amino acid composition and carbohydrate content of earthworm cuticle collagen and gelatin. A preparation of purified earthworm cuticle collagen proved to be a potent source of chemoattractant for garter snakes. Further, it was not possible to prepare chemoattractant from decuticlized earthworms. These results strongly suggest that a component of the earthworm chemoattractant for snakes is structurally related to earthworm cuticle collagen.Earthworms (Lumbricus terrestris) are a favorite prey of garter snakes of the genus Thamnophis and form a major component of their natural diet (1, 2). Garter snakes respond to objects coated with warm-water washings from earthworms by rapid tongue-flicking and an attack (1-3), thus suggesting that these washings contain a chemoattractant for the snake. In addition, garter snakes can be trained to follow trails of earthworm washings to receive bits of worm as rewards (4,5). Garter snakes use their vomeronasal systems, but apparently do not require a functional olfactory system, to detect and appropriately respond to this chemoattractant contained in earthworm washings (3,(6)(7)(8). Substances that stimulate the vomeronasal organs are typically naturally occurring complex large molecules that have been difficult to characterize chemically (9)(10)(11)(12)(13)(14).Earthworm washings (EWW), prepared as described by Wilde (3) and Burghardt (1), have been found to contain a chemoattractant for garter snakes that is water-soluble, absorbs at 280 nm, contains carbohydrate, and gives a positive reaction with ninhydrin and positive Lowry and Bradford tests for protein (15)(16)(17).A solution of EWW that had been dialyzed, lyophilized, and reconstituted in 0.15 M NaCl to 5 times its original concentration was separated on various calibrated gel-exclusion columns (16). When 0.15 M NaCl was used as eluent, two peaks were detected by 280 nm absorption (16). The first peak, F2, comprising components of Mr > 65,000, contained all the chemoattractant. This peak also contained neutral carbohydrate and ninhydrin-positive material and gave positive tests for protein. The second peak, F4, comprising components of much lower molecular weight, contained neutral carbohydrate but was devoid of chemoattractant (16).Snakes were able to discriminate the chemoattractant in EWW (pH 5.8) from solvent after the EWW was heated f...
