Characterization of vomeronasally-mediated response-eliciting components of earthworm wash-II

Garter snakes reliably spend more time and tongue flick more frequently at a dish containing surface washings of earthworm (EW) than at a control dish containing water or sodium chloride. In a series of experiments EW was examined to further characterize and purify the snake-attracting component(s). Earthworm wash boiled for up to two hours did not lose its snake-attracting activity, but EW boiled for four hours was not discriminated from a control substance. The active components of EW were completely precipitated by 90 and 100% saturated ammonium sulfate solutions. The molecular weight of the active components, as determined by dialysis, is larger than 67,000 daltons. Earthworm wash was separated into two peaks on either G-75 or AcA 44. The larger peak (F 2) contains snake-attracting material of molecular weight greater than 67K. The smaller peak material (F 4) is inactive as a snake-attractant and contains material of approximate molecular weight of 3K. Biological activity of EW covaried with Lowry (protein) assay and Dubois (carbohydrate) assay values and 280 nm absorbance. The response eliciting components of EW are not extractable from earthworms at temperatures below 60°C, but can be extracted at 60°C (for one minute) from the worms or the soil that forms the bedding for the earthworms.