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 PARIETALIS
LIFE 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 nonvolatile 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
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

http://www.crestock.com/image/476113-humanliver.aspx

Earthworm prey chemoattractant mechanism
still unknown
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
LATENCY TO ATTACK PROTOCOL

Present swab to acclimated snake
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 chemosensory
ability of HDX?

RESULTS: SWAB TRIAL WEEK 3
SHAM behavior fluctuates
 Consistent HDX non-biters
indicate deficit

RESULTS: SWAB TRIAL WEEK 5
Treatment numbers drop
due 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 secretion
collections to create
dilutions with an
unknown amount of
protein
PROTEIN DETERMINATION

Compare unknown dilutions to a standard curve of
known protein concentrations to determine unknown
concentrations
RESULTS: PROTEIN
DETERMINATION
Protein concentration ( g/ l)
P=0.027
8
6
4
2
0
SHAM
HDX
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 BabcockKrenk, Mattie Squire, Kata Haeberlin
