Isolation, identification, and biological characterization of the sea lamprey (Petromyzon marinus) migratory pheromone

This dissertation establishes the chemical identity, behavioral activity, and environmental origins and fates of a migratory pheromone employed by the sea lamprey (Petromyzon marinus), an ancient species of fish. Although previous studies had demonstrated that larval sea lamprey release an odor (pheromone) that guides adults to spawning streams, the complete composition of this odor and its biological activity were unknown. Initial tests using adsorbent resins revealed that XAD7HP was both effective and efficient at extracting and concentrating the pheromone from larval holding water. This resin was then employed to extract large quantities of the pheromone, which was subsequently purified using high pressure liquid chromatography (HPLC) and mass spectrometry (MS). Concurrent use of two bioassays (olfactory screening using electro-olfactogram recording from migratory adults and behavioral tests using 2-choice mazes supplied with lake water) led to the eventual isolation of three compounds with high biological activity. These three were: petromyzonol sulfate ('PS'; a compound previously suspected of having pheromonal activity), an unknown compound with a mass of 590.4 daltons, and a third unknown compound with a mass of 704.4 daltons. The third compound was the most active and had olfactory and behavioral activity at a concentration of 10-13 Molar (M). A mixture of all three compounds could account for most of the pheromone's behavioral activity. Collaborative studies employing nuclear magnetic resonance (NMR) established that both of the new compounds were novel disulfated steroids, which we identified and named petromyzosterol disulfate ('PSDS'), and petromyzonamine disulfate ('PADS'). Subsequent analyses of larval holding waters and stream waters using MS showed that larval sea lamprey release ∼10 to 30 ng/larva/hr of all three compounds and that PS and PADS are present in river waters at biologically relevant concentrations. We also found that these compounds have a half-life of ∼3 days in natural waters. Together, these studies demonstrate that PADS, PSDS, and PS constitute the majority of the sea lamprey migratory pheromone, the first migratory pheromone identified in a vertebrate. A mixture of these compounds clearly has promise for use controlling sea lampreys in the Great Lakes where they are a serious invasive pest.