Chemical communication by pheromones is the predominant mode of information transfer in most vertebrates. The female garter snake (Thamnophis sirtalis) sex pheromone, a nonpolar, hydrophobic blend of saturated and monounsaturated methyl ketones, is insoluble in aqueous solutions. This pheromone is detected by the vomeronasal organ (VNO), which is specialized for the reception of nonvolatile chemical cues, but the mechanism by which the pheromone gains access to the aqueous environment of the organ is unknown. Preliminary data indicate that the Harderian glands' (HG) secretions, which duct exclusively into the VNO in snakes, contain solubilizing and/or pheromone-binding properties. For over 300 years, the function of the cephalic HG has been the subject of speculation from numerous sources. Certainly their relatively large size, their phylogenetic age, and persistent conservation in almost all tetrapod groups as they emerged from an aqueous to an air/land environment suggest that they probably play an important role in the physiological adaptation to terrestrial life. This project will examine the role of the HG as a mediator in providing access for the female sex pheromone to the VNO of male garter snakes. Using radiolabeled garter snake sex pheromone, studies will be conducted to determine the solubilizing effect of HG secretion on the female sex pheromone and the potential of the HG secretion to possess pheromone-binding proteins. The effects of HG removal on courtship behavior and prey attack, both known to require a functional VN system, will also be investigated, as will the effects of seasonal and hormonal variation on the composition of the HG. The integrative research described here will provide demonstrations of a novel mechanism by which nonpolar, hydrophobic pheromone molecules reach the VNO, using a very well characterized system: the VN system of garter snakes. If a pheromone-binding protein is involved, it will be the first identified in a non-mammalian species. This research will provide a compelling model for the examination of HG involvement in perireceptor events mediating VNO function of other mammalian and non-mammalian vertebrates. In addition, this study takes advantage of two unambiguous, quantifiable behaviors exhibited by a species in its natural habitat that not only offers a robust model for insights into seasonal shifts in reproductive behavior and feeding, but also proposes a physiological mechanism mediating these seasonally regulated changes. This proposal involves an integrative, multidisciplinary approach including field and lab studies that will yield significant insights into the unifying principles of pheromonal communication common to many, if not most, vertebrates. The broader impacts of this proposal include multidisciplinary training in research and education through the mentoring of graduate students, undergraduates and outreach to high school and grade school students. Undergraduates are an integral part of the PI's laboratories, assisting in animal husbandry, field work and behavior experiments. These undergraduates have been co-authors on 23 published papers from the two laboratories. The PI's have a long-term commitment to mentoring, with more than a dozen high school students involved in minority training programs and Intel/Westinghouse competitions, and an additional nine high school students having worked in an NSF-initiated Apprenticeships in Science and Engineering program. These experiences, coupled with appropriate course work, will provide a firm foundation for these scientists-in-training to experience the joys and frustration of field and laboratory work.
