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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
THE HARDERIAN GLAND
Critical role in garter snake chemoreception Regeneration ability
THAMNOPHIS SIRTALIS PARIETALISLIFE HISTORY
Warm summer months – feeding
Early fall – prepare for winter hibernation
Early spring – breeding
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 proteinNC=nasal cavity
VNO=vomeronasal organ
HG=Harderian gland
LC=lacrimal canal
LD=lacrimal duct
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
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
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
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
MY RESEARCH
Question: Does a relationship exist between the rate of Harderian Gland regeneration and chemosensory recovery in red-sided garter snakes?
My Research
Hypotheis: The Harderian Gland plays a critical role in chemoreception of red-sided garter snakes and regeneration is correlated
to chemosensory ability.
EXPERIMENTAL DESIGN
Purpose Establish a deficit in chemosensory ability using
behavioral and biochemical assays Track HG regeneration through time using
histology Treatments
HDX SHAM
EXPERIMENTAL DESIGN
Two Protocols to establish deficit Latency to attack – behavioral assay Protein determination – biochemical assay
Photo by Don Powers
LATENCY TO ATTACK PROTOCOL Collect earthworm shock secretion for swab
Present swab to acclimated snake
LATENCY TO ATTACK PROTOCOL
LATENCY TO ATTACK PROTOCOL
Time from first tongue flick until bite or to a max of 30 seconds (indicates no bite)
RESULTS: SWAB TRIAL WEEK 1
Significant difference in bite times (P = 0.025)
RESULTS: SWAB TRAIL WEEK 2
Non-biting SHAMs can be attributed to other factors
Recovered chemosensoryability of HDX?
RESULTS: SWAB TRIAL WEEK 3
SHAM behavior fluctuates Consistent HDX non-biters
indicate deficit
RESULTS: SWAB TRIAL WEEK 5
Treatment numbers dropdue to perfusions
Consistent non-biters remain
RESULTS: SWAB TRIAL WEEK 8
PERFUSIONS/HISTOLOGY
Perfusion performed weekly to preserve Harderian gland tissue for histology
PROTEIN DETERMINATION
Quantify a difference in protein concentration of HG secretions between HDX and SHAM treatment groups
HG SECRETION COLLECTION
Collect HG secretions Pilocarpine
Use secretioncollections to createdilutions with anunknown amount of protein
PROTEIN DETERMINATION
Compare unknown dilutions to a standard curve of known protein concentrations to determine unknown concentrations
RESULTS: PROTEIN DETERMINATION
SHAM HDX
Pro
tein
con
cent
ratio
n (
g/ l
)
0
2
4
6
8
P=0.027
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)
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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