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