1. SECRETS OF CHEMORECEPTION: REGENERATION OF THE HARDERIAN GLAND KING YABUT DR. ROBERT T. MASON DEPARTMENT OF ZOOLOGY OREGON STATE UNIVERSITY

2. T HE H ARDERIAN G LAND Discovered by Johann Jacob Harder in 1694 Found in virtually all land vertebrates Function remains largely unknown

3. T HE H ARDERIAN G LAND Critical role in garter snake chemoreception Regeneration ability

4. T HAMNOPHIS SIRTALIS PARIETALIS L IFE H ISTORY Warm summer months – feeding Early fall – prepare for winter hibernation Early spring – breeding

5. T HE V OMERONASAL S YSTEM AND THE R OLE OF S EMIOCHEMICALS Chemoreception is crucial to garter snake behavior The VN system is used for detection of non- volatile semiochemicals Female attractiveness sex pheromone Prey chemoattractants – earthworm shock secretion protein NC=nasal cavity VNO=vomeronasal organ HG=Harderian gland LC=lacrimal canal LD=lacrimal duct

6. T HE R OLE OF THE H ARDERIAN G LAND Female attractiveness sex pheromone is a lipid VNO environment is aqueous Harderian Gland is the major source of fluid for the VNO Dyes injected into the HG suggest link to VN system NC=nasal cavity VNO=vomeronasal organ HG=Harderian gland LC=lacrimal canal LD=lacrimal duct

7. T HE R OLE OF THE H ARDERIAN G LAND Lipid binding proteins from Harderian Gland Homogenate of HG solubilized sexual attractiveness pheromone Harderianectomized males do not court females

8. PREVIOUS RESEARCH FROM MASON LAB HDX snakes also show a loss in feeding behaviors Chemosensory ability began to progressively return indicating a crucial role in feeding behavior

9. PREVIOUS RESEARCH FROM MASON LAB Study links HG to feeding behavior Previously unknown regeneration ability indicates importance Earthworm prey chemoattractant mechanism still unknown http://www.crestock.com/image/476113-human- liver.aspx

10. MY RESEARCH Question: Does a relationship exist between the rate of Harderian Gland regeneration and chemosensory recovery in red-sided garter snakes?

11. My Research Hypotheis: The Harderian Gland plays a critical role in chemoreception of red-sided garter snakes and regeneration is correlated to chemosensory ability.

12. EXPERIMENTAL DESIGN Purpose Establish a deficit in chemosensory ability using behavioral and biochemical assays Track HG regeneration through time using histology Treatments HDX SHAM

13. EXPERIMENTAL DESIGN Two Protocols to establish deficit Latency to attack – behavioral assay Protein determination – biochemical assay Photo by Don Powers

14. LATENCY TO ATTACK PROTOCOL Collect earthworm shock secretion for swab

15. Present swab to acclimated snake L ATENCY TO A TTACK P ROTOCOL

16. Time from first tongue flick until bite or to a max of 30 seconds (indicates no bite)

17. RESULTS: SWAB TRIAL WEEK 1 Significant difference in bite times ( P = 0.025)

18. RESULTS: SWAB TRAIL WEEK 2 Non-biting SHAMs can be attributed to other factors Recovered chemosensory ability of HDX ?

19. RESULTS: SWAB TRIAL WEEK 3 SHAM behavior fluctuates Consistent HDX non-biters indicate deficit

20. RESULTS: SWAB TRIAL WEEK 5 Treatment numbers drop due to perfusions Consistent non-biters remain

21. RESULTS: SWAB TRIAL WEEK 8

22. PERFUSIONS/HISTOLOGY Perfusion performed weekly to preserve Harderian gland tissue for histology

23. P ROTEIN D ETERMINATION Quantify a difference in protein concentration of HG secretions between HDX and SHAM treatment groups

24. HG S ECRETION C OLLECTION Collect HG secretions Pilocarpine Use secretion collections to create dilutions with an unknown amount of protein

25. P ROTEIN D ETERMINATION Compare unknown dilutions to a standard curve of known protein concentrations to determine unknown concentrations

26. RESULTS: PROTEIN DETERMINATION

27. CONCLUSION Behavioral assay Inconsistency in SHAM behavior may be due to other factors Consistent HDX non-biters may indicate a deficit in chemosensory ability Results are incomplete until correlated with histology Protein determination Significant statistical difference between SHAM and HDX protein concentrations ( P = 0.027)

28. ACKNOWLEDGEMENTS Dr. Robert T. Mason Dr. Kevin Ahern and the Howard Hughes Medical Institute Chris Friesen Rocky Parker Mason Lab –Ben Burke, Miranda Babcock- Krenk, Mattie Squire, Kata Haeberlin