1. Effects of UVB radiation on the skin lipids of garter snakes Anna Vigeland Dr. M. Rockwell Parker Dr. Robert T. Mason

2. Study Species Red-sided Garter Snake (Thamnophis sirtalis parietalis) Model organism Hibernate 8 months in large hibernacula Emerge by the thousands in late spring Breeding follows for a few weeks Courtship behavior mediated by pheromones produced in the skin

3. Garter Snake Environment Most northerly living reptile Study snakes caught at dens in Manitoba Northern regions strongly subject to environmental changes UVB radiation

4. Skin Lipid Profile 18 long-chain saturated and monounsaturated methyl ketones in pheromone High ratios of unsaturated and high molecular weight methyl ketones are associated with large, healthy females Males differentiated in part by the presence of squalene more attractive less attractive

5. Skin Lipid Profile Gas chromatograph of a typical female pheromone sample Saturated Unsaturated 394 408 420 422 434 436 492 478 450 464 506 448 462 476 490 504 518 532

6. Objective Methyl ketone ratios change during the summer in the field but not in the lab Why?

7. Hypothesis Ultraviolet radiation causes qualitative changes in the skin lipid profiles of red-sided garter snakes

8. Question 1 Does ultraviolet-B radiation have an effect on the female sexual attractiveness pheromone?

9. Methods

10. UVB radiation on female pheromone samples Full-spectrum light onlyFull-spectrum and UVB light

11. * * * * * * * * p < 0.05

12. UVB exposure affects the ratios of pheromone samples p = 0.00934

13. Question 2 Does ultraviolet-B radiation have the same effect on the female sexual attractiveness pheromone when in mixture with other skin lipids?

14. Methods

15. Pheromone irradiated within neat solution Full spectrum light only Full spectrum and UVB light

17. Pheromone irradiated within neat solution

18. Two groups of snakes exposed to either full spectrum or full spectrum +UVB light Behavioral and physical changes examined after prolonged UV exposure Skin lipid analysis after exposure period Live-animal exposure: overview

19. Question 3 Does exposure to ultraviolet-B radiation have an effect on the behavior of red-sided garter snakes?

20. Methods Short-term: detection and/or avoidance of UVB light Cardboard tube test: % snakes seeking refuge in tubes Mylar filter test: % snakes situated under Mylar vs. acetate Long-term: snake activity levels Observation and categorization of behaviors: % snakes active, alert, or resting

21. UVB radiation does not affect snake behavior No significant differences in number of snakes seeking refuge in cardboard tubes Equal numbers of snakes situated under Mylar filters as under acetate filters No significant differences in the activity levels between treatment groups or after the 15-day exposure period

22. Question 4 What effects does daily exposure to ultraviolet-B radiation have on the neutral skin lipids of red-sided garter snakes?

23. Methods Skin lipid extraction by soaking in hexane Purification of female attractiveness pheromone and squalene by alumina column chromatography Analysis of pheromone concentrations by gas chromatography

26. Daily UVB on live snake pheromone ratios

27. Environmental Implications UVB radiation has an effect on the female attractiveness pheromone and its US:S ratio UVB radiation has been increasing, especially in polar latitudes, due to ozone depletion Increases in UVB could affect red- sided garter snake reproductive behavior

28. Further Research Gas chromatography analysis of squalene concentrations Thin layer chromatography to test for major differences in other neutral skin lipids

29. Acknowledgements Funding provided by: Howard Hughes Medical Institute Special thanks to: Dr. Robert T. Mason Dr. M. Rockwell Parker Mason Lab: Emily Uhrig, Mattie Squire, Chris Friesen, Ben Burke The Blaustein Lab

30. Questions?