An unresolved question in animal behavior is how complexity and variation in male signals is maintained within populations. Past attempts to identify evolutionary forces that result in different male reproductive signals have ignored the possibility that particular trait combinations may gain effectiveness by the synergistic interaction of individual signal components. In fact, these interactions may be a vital component of selection on complex male displays. This study will focus on pheromones in a species of salamander. These reproductive pheromones are chemical signals, created by a combination of proteins, that a male delivers to a female during courtship. The combination of individual pheromone components makes each male unique. Behavioral experiments will measure the strength of female preferences for particular pheromone combinations. Documenting these female preferences will reveal whether this mechanism of female mate choice can account for the immense variation of male pheromones. Additionally, mathematical models of the process by which pheromone diversity is maintained will be constructed, and evaluated using the experimental results.
This study will promote outreach programs at Oregon State University and at the University of Louisville. These programs will link faculty and students (graduate and undergraduate) with local teachers (K-12) and with students traditionally under-represented in scientific research. The three primary goals of this activity are to train graduate and undergraduate students in experimental approaches and in new techniques, to put into practice new teaching methods shown to promote learning and retention in large science courses, and to let faculty and graduate students work with local school teachers to develop short programs amenable to different grade levels.
About 100 million years ago (MYA), terrestrial, lungless plethodontid salamanders evolved a unique chemical communication system in which a courting male vaccinates a female with a pheromone produced by a gland on the male’s chin. About 20 MYA, some plethodontid species developed an alternate slapping method by which a male delivers protein pheromones directly to the female nares in order to stimulate areas of the brain that increase her sexual receptivity. Because of the direct path from gene to protein to behavior, the plethodontid courtship system provides exceptional interdisciplinary research opportunities. In the salamander, P. shermani, which utilizes slapping delivery, we have previously identified two major families of pheromones that are annually produced in a male gland in order to modulate female reproductive behavior. As part of a collaborative multi-university team, we are now investigating: how the pheromone genes are specifically regulated at the DNA level; how the information contained in those genes is translated, in a highly regulated process, into mature pheromone proteins; how the gland develops at a specific time each year and stores pheromones until the male delivers them to the female; and, how the effects of these pheromones enhance female sexual receptivity. The results of our collaborative studies have broad interdisciplinary impact and indirect biomedical relevance with respect to transcriptional and translational regulation. In earlier work, we purified and characterized Plethodontid Receptivity Factor (PRF; related to IL-6 cytokines) and showed that either native or an identical synthetic PRF alone could increase female receptivity. Recently, we prepared a synthetic Plethodontid Modulating Factor (PMF; related to snake cytotoxins and several human proteins) that is biochemically identical to the most abundant natural PMF and assayed its biological effects. Using the molecular probe, agmatine, we previously demonstrated that sensory neurons in the female are activated by male courtship pheromones and extended that work to demonstrate neural effects at sites in the brain (amygdala, preoptic area, and hypothalamus) associated with pheromone reception in other vertebrates. These studies formed the basis for ongoing molecular studies of potential pheromone receptors in the P. shermani VNO. We determined that our protein pheromones PRF and PMF have undergone rapid evolutionary shifts in composition. Biochemical analyses and DNA sequencing revealed extraordinary hypervariability. An individual male can produce only 3 PRFs, but at least 30 PMF isoforms. Also, PMF is produced by a multigene family characterized by many gene duplications. This hypervariability can be explained in terms of our working hypothesis: males vary in pheromone isoforms, female response to certain isoforms changes over time, and the expression of male pheromone isoforms is altered as an adaptive evolutionary response to changes in female preference. Ongoing experiments that will result in future publications that partially credit this grant include: (A) histological and immunohistochemical analyses of the developing mental gland in samples collected at 3-week intervals as the gland annually develops (May-July) and converts to a pheromone factory during the courtship season (Aug-Sept); (B) transcriptome analyses for selected developmental time points. Thus far, a preliminary bioinformatics analysis has been performed for 6 billion bases and is providing intriguing information that will shape future experiments related to the possible roles of environmental variables in gland development and pheromone production. BROADER IMPACTS We participated in the typical and important training of undergraduate and graduate students. Two graduate students and the senior research technologist who manages our lab (all supported by this grant or an NSF-GRF) routinely participated in the training of undergraduates on individual projects relating to the molecular characterization of plethodontid pheromones in three complementary salamander species. One of the graduate students, served as an assistant coach to a high school Science Olympiad team (at the state and national level), applying the knowledge and experience gained as an undergraduate and graduate student to help educate high school students in several disciplines including protein modeling, cell biology, ecology, and experimental design. The other graduate student completed her M.S. in Biochemistry (May, 2012) and decided to pursue the teaching of science at either the middle school or high school level. She entered the University of Louisville (UofL) M.A.T program as an NSF Noyce Scholar in May 2012, will graduate in Aug, 2013 and is expected to be an excellent teacher and role model. Our senior research technologist completed her M.A.T. in May, 2012 and plans to teach high school chemistry at some future point. In addition, the Co-PI, PW Feldhoff, as the UofL Project Director of the NSF KY-WV LSAMP grant continued: to give talks to minority groups about STEM area opportunities; to meet individually with LSAMP-supported students; organized and directed UofL undergraduate summer research programs that included an annual student research day to which non-scientists were invited; and, participated at many meetings that have the potential to increase awareness of the opportunities and benefits of undergraduate research.
