**ABSTRACT NOT FOR CITATION WITHOUT AUTHOR PERMISSION. The title, authors, and abstract for this completion report are provided below.  For a copy of the full completion report, please contact the author via e-mail at KuhnL@msu.edu. Questions? Contact the GLFC via email at frp@glfc.org or via telephone at 734-662-3209.**


 Development of antagonists to pheromonal sulfated steroids for sea lamprey control


 1-4Leslie A. Kuhn, 2Sebastian Raschka, 3Anne M. Scott, and 3Weiming Li


 1Department of Biochemistry & Molecular Biology Michigan State University 502C Biochemistry Building, 603 Wilson Road East Lansing, MI 48824


2Department of Computer Science & Engineering Michigan State University East Lansing, MI 48824


3Department of Fisheries & Wildlife Michigan State University 142 Giltner Hall, 293 Farm Lane East Lansing, MI 48824


December 2017




The attraction of female sea lampreys to spawning areas, via pheromones released by nesting males, is a strong, evolved response that other control approaches address by creating barriers or traps to reduce upstream movement. The goal of our research was to tackle the problem at a fundamental level by negating the nest attraction force. In previous GLFC-supported research, we identified two compounds that neutralize both the olfactory potency of 3kPZS, the principal mating pheromone released by male sea lamprey, and its behavioral effects in ovulated females, by integrating hypothesis-driven computational screening and electro-olfactogram and behavioral assays. Here, we have tested the hypothesis that  compounds identified by computational screening that specifically mimic DkPES (3,12-diketo-4,6-petromyzonene-24-sulfate) and PAMS-24 (3β,5α,7α,24R)-1-[3-[[24-sulfooxy-cholestan-3-yl]amino]-propyl]-2-pyrrolidinone), two additional components of the mating pheromone released by male sea lamprey, will reduce their olfactory detection and provide synergistic tools for controlling sea lamprey behavior. As described in the publications in the Appendix, this research necessitated the development of several novel computational methods for the discovery of pheromone antagonists for sea lamprey control, with future applicability to other invasive species and inhibitor discovery projects: Screenlamp, which enables high-throughput screening of millions of commercially available small organic molecules as pheromone (or other bioactive molecule) mimics, including hypothesis-based filtering on the presence or absence of matches to specific chemical groups in the pheromone; SiteInterlock, which uses rigidity  theory to predict the native mode of binding between small organic molecules, including pheromones, and

their receptor protein structures; CholMine, which predicts how bile acids, including sea lamprey pheromones and cholesterol-like molecules, interact with their receptors, by using interaction patterns derived from experimental data from the Protein Data Bank; and a suite of data mining protocols designed to identify the presence of specific chemical groups that discriminate between active and inactive compounds based on analyzing the biological activity values and 3D chemical structures of the assayed molecules. Following identification of pheromone mimics by computational screening, biological testing of these potential antagonists of 3kPZS, DkPES, and PAMS-24 was performed in three tiers. First, electro-olfactogram (EOG) assays were used to measure the extent to which each compound identified by screening reduced the sea lamprey olfactory response to the corresponding pheromone. Next, the subset of the compounds that were most active in reducing pheromone olfaction were tested in a two-choice maze to assess the behavioral preference for the treatment or control (vehicle) side of the maze. Preference for the treatment or control side was evaluated for ovulated female sea lamprey based on the

time spent in each side. Finally, for those compounds that reduced the preference or repelled the ovulated female sea lampreys from the pheromone (3kPZS, DkPES, or PAMS-24), in-stream assays were performed to test the behavioral response in a natural stream. Pheromone was applied at a simulated nest, and pheromone plus antagonist molecule(s) were applied in a neighboring simulated nest. The behavioral assays show that PZS (petromyzonol sulfate) and 3sPZS (trisulfated PZS) have synergistic activity as antagonists. This combination in the upper Ocqueoc River was more effective in reducing upstream movement, entry to the 3kPZS-baited simulated nest, and retention at the 3kPZS-baited nest when compared to the application of each antagonist alone. Similarly, while spermiated male washings (SMW), containing the entire complement of male mating pheromone and normalized to a specific concentration of 3kPZS, were highly attractive to ovulated female sea lampreys in the two-choice maze, SMW combined with a similarly low concentration of PZS and 3sPZS in the same maze proved to be repulsive to ovulated females. Based on these results, our integrative approach has identified a combination of pheromone antagonists that nullify the pheromone olfactory and behavioral responses and show potential for sea lamprey control, by blocking the nest attraction needed for reproductive success.