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SECRETS OF CHEMORECEPTION: REGENERATION OF THE HARDERIAN GLAND

SECRETS OF CHEMORECEPTION: REGENERATION OF THE HARDERIAN GLAND. KING YABUT DR. ROBERT T. MASON DEPARTMENT OF ZOOLOGY OREGON STATE UNIVERSITY. The Harderian Gland. Discovered by Johann Jacob Harder in 1694 Found in virtually all land vertebrates Function remains largely unknown.

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SECRETS OF CHEMORECEPTION: REGENERATION OF THE HARDERIAN GLAND

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

  2. The Harderian Gland • Discovered by Johann Jacob Harder in 1694 • Found in virtually all land vertebrates • Function remains largely unknown

  3. The Harderian Gland • Critical role in garter snake chemoreception • Regeneration ability

  4. Thamnophis sirtalisparietalisLife History Warm summer months – feeding Early fall – prepare for winter hibernation Early spring – breeding

  5. The Vomeronasal System and the Role of Semiochemicals • 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. The Role of the Harderian Gland • 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. The Role of the Harderian Gland • 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. Latency to Attack Protocol • Present swab to acclimated snake

  16. Latency to Attack Protocol • 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. Protein Determination • Quantify a difference in protein concentration of HG secretions between HDX and SHAM treatment groups

  24. Collect HG secretions Pilocarpine Use secretion collections to createdilutions with an unknown amount of protein HG Secretion Collection

  25. Protein Determination • 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

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