AN ABSTRACT OF THE THESIS OF
Anna E. Vigeland for the degree of Honors Baccalaureate of Science in Biochemistry and
Biophysics presented on May 31, 2011. Title: UVB Radiation Affects the Quality of the
Female Sexual Attractiveness Pheromone of the Red-Sided Garter Snake.
Abstract approved:
Robert T. Mason
In this study, pheromone and skin lipid samples collected from red-sided garter
snakes (Thamnophis sirtalis parietalis) were exposed directly to either full-spectrum light
or full-spectrum plus UVB light. In addition, female and male snakes were exposed to
daily doses of either full-spectrum or full-spectrum and UVB light, after which the skin
lipids were collected and analyzed. Behavioral studies were conducted on the snakes
during exposure. After each experiment, the pheromone and other lipids were weighed
and qualitatively and quantitatively analyzed by gas chromatography and mass
spectrometry. The results show that UVB radiation reduces the unsaturated:saturated and
the high-molecular weight:low-molecular weight methyl ketone ratios of the pheromone,
which are correlated with the level of attractiveness of a female snake, in directly
exposed samples. The unsaturated:saturated ratio was also decreased in live female
snakes. No behavioral differences were observed, indicating that snakes do not attempt
to shelter themselves from UVB light and appear to be unaware of UVB differences.
These results suggest that an increase in UVB radiation, as has been occurring in the redsided garter snakes' native range in Canada, could have an effect on the mating behavior
of the snakes, leading to changes in the timing of critical life-history events such as
breeding and dispersal for feeding.
Key Words: snake, Thamnophis sirtalis parietalis, UVB, lipid, pheromone
Corresponding email address: vigeland@hawaii.edu
©Copyright by Anna E. Vigeland
May 31, 2011
All Rights Reserved
UVB Radiation Affects the Quality of the Female Sexual Attractiveness Pheromone
of the Red-Sided Garter Snake
by
Anna E. Vigeland
A PROJECT
submitted to
Oregon State University
University Honors College
in partial fulfillment of
the requirements for the
degree of
Honors Baccalaureate of Science in Biochemistry and Biophysics (Honors Scholar)
Presented on May 31, 2011
Commencement June 2011
Honors Baccalaureate of Science in Biochemistry and Biophysics project of Anna E.
Vigeland presented on May 31, 2011
APPROVED:
________________________________________________________________________
Mentor, representing Zoology
________________________________________________________________________
Committee Member, representing Biochemistry and Biophysics
________________________________________________________________________
Committee Member, representing Zoology
________________________________________________________________________
Dean, University Honors College
I understand that my project will become part of the permanent collection of Oregon
State University, University Honors College. My signature below authorizes release of
my project to any reader upon request.
________________________________________________________________________
Anna E. Vigeland, Author
Acknowledgements
Firstly, I would like to extend a great appreciation to my mentor, Bob Mason. He
has offered me much insight into the scientific process and advice on writing a research
report. The experience he has given me in his laboratory has been and will be invaluable
in my career as a researcher.
Secondly, I would like to thank Rocky Parker, who acted as my mentor during
Bob’s sabbatical. He is responsible for training me in all the laboratory protocol I used
during this research and for getting the project started. This thesis has been made
possible thanks to him.
Next, I would like to offer my appreciation to Emily Uhrig and to Chris Friesen,
who have provided the assistance and support needed to keep the project running and to
keep me going the right direction. My fellow undergraduate researcher, Mattie Squire,
has also been a great person to work alongside throughout our time in the lab. A huge
thanks needs to be given to Ben Burke, who was responsible for the feeding, cleaning,
and other care of all of the snakes.
I would also like to thank the members of the Blaustein Laboratory, especially
Catherine Searle and Andrew Blaustein, for permitting me to use their UV facility and
equipment, and for consulting with us on using UV radiation in research.
Finally, I would like to express my profound appreciation for Kevin Ahern, the
best academic advisor I could have asked for, and Indira Rajagopal, for teaching me how
to be a great scientist.
Funding for this project was provided by the Howard Hughes Medical Institute.
Table of Contents
Introduction……………………………………………………………………....……….1
Materials and Methods……………………………………………………………………5
Ultraviolet B Exposure……………………………………………………………5
Isolated Pheromone Experiment…………………………………………………..6
Neat Solution Experiment…………………………………………………………6
Snake Care………………………………………………………………………...7
Behavior Tests……………………………………………………………………..8
Live-exposure Lipid Samples……………………………………………………...9
Column Chromatograhy/Mass Spectrometry……………………………………10
Gas Chromatography……………………………………………………………10
Data Analysis…………………………………………………………………….11
Results……………………………………………………………………………………12
Isolated Pheromone Experiment…………………………………………………12
Neat Solution Experiment………………………………………………………..17
Behavior Studies…………………………………………………………………23
Live-exposure Lipid Samples…………………………………………………….25
Discussion………………………………………………………………………………..31
References………………………………………………………………………………..33
List of Figures
1. Gas chromatogram of a typical female pheromone profile……………………………2
2. Gas chromatogram of an exemplary female pheromone sample, the half exposed
to full-spectrum light for 80 hours……………………………………………….12
3. Gas chromatogram of the exemplary female pheromone sample, the half exposed
to full-spectrum and UVB light for 80 hours………………………...…………..13
4. Average concentrations in solution of the 18 individual methyl ketones in female
isolated pheromone samples……………………………………………………..14
5. Unsaturated:saturated and high molecular weight:low momecular weight methyl
ketone ratios in female isolated pheromone samples…………………………….15
6. Average concentrations in solution of the 18 individual methyl ketones in male
isolated pheromone samples……………………………………………………..16
7. Gas chromatogram of an exemplary male pheromone sample, purified from the
half of the neat solution exposed to full-spectrum light for 80 hours……………18
8. Gas chromatogram of the exemplary male pheromone purified from the half of
the neat solution exposed to full-spectrum and UVB light for 80 hours………...18
9. Average concentrations in solution of the 18 individual methyl ketones in
female skin lipid samples………………………………………………………...19
10. Unsaturated:saturated and high molecular weight:low molecular weight methyl
ketone ratios in female skin lipid samples…………………………………...…..20
11. Average concentrations in solution of the 18 individual methyl ketones in male
skin lipid samples………………………………………………………………...21
12. Average concentrations in solution of the major lipids in female skin lipid
samples…………………………………………………………………………..22
13. Average concentrations in solution of the major lipids in male skin lipid samples...23
14. Percentage of snakes in each treatment group participating in one of three
behavior types each day for the first 7 days of exposure……………….………..24
15. Percentage of snakes in each treatment group participating in one of three
behavior types each day for the last 7 days of exposure…………………….…..25
16. Average skin surface concentrations of the 18 individual methyl ketones in liveexposure female pheromone samples………………………………………...….26
17. Unsaturated:saturated methyl ketone ratios in the skin lipids of female snakes…….27
18. Average skin surface concentrations of the major lipids in live-exposure skin
lipid samples from female snakes………………………………………………..28
19. Average skin surface concentrations of the 18 individual methyl ketones in liveexposure male pheromone samples……………………………………………...29
20. Average skin surface concentrations of the major lipids in skin lipid samples
from male snakes………………………………………………………………...30
Introduction
The ability to locate, identify, and attract potential mates is critical to an animal’s
reproductive fitness and success in coordinating reproductive behaviors. Signals used to
convey information about an individual such as species, sex, state of health and
reproductive maturity come in many forms, such as visual, auditory, or chemical cues
(Darwin, 1871). Pheromones are chemicals secreted externally from an organism that
carry information from one individual of a species to another (Karlson and Lüscher,
1959), and have been widely studied in a variety of insects (Butler, 1967) including
moths (Shorey, Gaston, and Fukuto, 1964), bees (Boch, Shearer, and Stone, 1962), and
termites (Moore, 1966) and to some extent in vertebrate species including tree frogs
(Wabnitz et. al., 1999), elephants (Rasmussen et. al., 1997), and goldfish (Dulka et. al.,
1987). Very few reptilian pheromones have been characterized, the best studied being
that of the red-sided garter snake (Thamnophis sirtalis parietalis) which is secreted in the
skin of females to elicit courtship behavior from males (Mason et. al, 1989).
The red-sided garter snake is a subspecies of the common garter snake
(Thamnophis sirtalis) in the family Colubridae (Say, 1823).
Thamnophis sirtalis is
widespread across North America with parietalis occupying the northern portion of the
range, being found across Canada into its northern regions. As such, the red-sided garter
snake is the most northerly living reptile in the Western hemisphere and possibly the
world (Conant, 1975). Due to cold temperatures at these high latitudes, red-sided garter
snakes spend eight months out of the year in hibernation in large communal dens known
as hibernacula. In mid-April the snakes emerge from hibernation and exit the hibernacula
by the thousands to begin breeding.
Males emerge first and congregate at the
2
den site, followed by the females which emerge a few at a time over a period of 4-5
weeks (Gregory, 1974).
Mating primarily occurs at the den site immediately after
emergence, and once the breeding period is over the snakes disperse to begin feeding
over the summer (Garstka, Camazine, and Crews, 1982).
The female sexual attractiveness pheromone is a blend of nonvolatile saturated
and monounsaturated (ω-9) methyl ketones of varying length (C29-C37) (Mason et. al.,
1989). These are sequestered along with other lipids from the skin on the snakes’ dorsal
surface and picked up by other snakes by the tongue and transferred to the vomeronasal
organ in the roof of the mouth (Noble, 1937).
Figure 1. Gas chromatogram of a typical female pheromone profile. Each methyl
ketone peak is labeled by its molecular weight (Da) as unsaturated (red, italic) or
saturated (blue, bold).
3
Very little pheromone, on the order of micrograms or less, is required to entice a
male to exhibit robust courtship behavior, and males will even display courtship behavior
(increased tongue-flicking, chin-rubbing) towards isolated pheromone samples in the
absence of females (Mason and Crews, 1985).
The level of attractiveness of a
pheromone sample, measured by the level of courtship behavior elicited from a male, is
proportional to the ratios of unsaturated:saturated methyl ketones (hereafter referred to as
US:S) and high-molecular weight:low-molecular weight methyl ketones (being the 9
methyl ketones with the greatest molecular mass and the 9 methyl ketones with the
lowest molecular mass, respectively; hereafter referred to as HMW:LMW) in the sample.
Higher US:S and HMW:LMW ratios tend to be produced by larger females in better
condition, which are more reproductively fit (LeMaster and Mason, 2002).
Interestingly, male snakes also produce the female sexual attractiveness
pheromone, although at lower concentrations and with lower US:S and HMW:LMW
ratios than females. However, male snakes do not usually court other males, which is
thought to be due in part to the presence of squalene on male skin. Squalene is more
abundant in male skin lipid samples than female samples, and squalene added to female
pheromone samples reduces its attractiveness (Mason et. al., 1989).
A previously conducted study found that the pheromone from female red-sided
garter snakes kept in outdoor arenas during the two weeks of the study in Manitoba had
qualitative differences from newly emerged females, and that male courtship response to
these females diminished during this time.
This was hypothesized to be due to
environmental conditions that the snakes encounter naturally post-emergence (Uhrig et.
al., in review). One possible contributing environmental factor could be ultraviolet light
4
that the snakes experience above-ground. A study done on Cope’s rat snakes found that
ultraviolet-B (UVB) radiation inhibits catalase and superoxide dismutase and causes lipid
peroxidation in the snake skin (Chang and Zheng, 2003), so it is possible that UVB
radiation may be photochemically breaking-down the methyl ketones of the red-sided
garter snake pheromone. Squalene is also known to be peroxidized by exposure to UVB
radiation (Dennis and Shibamoto, 1989).
Ambient levels of ultraviolet-B type radiation have been increasing with the
depletion of ozone in the stratosphere, especially in the polar regions of the globe (Kerr
and McElroy, 1993). This rise in UVB levels has been linked to physiological stress in
aquatic ecosystems (Bancroft, Baker, and Blaustein, 2007), genetic instability in plants
(Ries et. al., 2000), and is believed to be a factor contributing to amphibian population
decline (Kiesecker, Blaustein, and Belden, 2001). If UVB radiation does have an effect
on the female sexual attractiveness pheromone, these increasing levels could have a
critical impact on the mating behavior of the red-sided garter snake. This thesis explores
the hypothesis that exposure to ambient levels of UVB radiation chemically alters the
female sexual attractiveness pheromone, leading to significant impacts on the
reproductive biology of this species in the field.
5
Materials and Methods
Ultraviolet B Exposure
UVB exposure in the project was conducted in a UV(-)use room in the Blaustein
Laboratory at Oregon State University. Lighting systems were set up in the room, which
had each section of shelves partitioned from the rest of the room by black plastic
sheeting. Observation holes approximately 10” by 6” had been cut in the sheets for
animal behavior studies. 10 gallon glass tanks were placed on the shelving below the
lights. To ensure full exposure of snakes to the light, each tank was furnished with only a
rock for shedding and a water dish. All tanks were exposed to approximately equal levels
of full-spectrum light, while UVB bulbs emitting light at a wavelength of 313 nm were
placed over only the tanks on one half of the shelves. UVB levels were measured using a
Solar Light Co. PMA 2100 spectroradiometer measuring at 313 nm. Average UVB
levels at the bottom of the snake tanks were calculated by placing the radiometer in the
front, back, and center of each tank and averaging the readings. Levels from the tops of
the tanks were determined by placing the radiometer on the top center of the tanks, in the
same position where the sample beakers were placed. For the Mylar filter test, levels
were measured by taking readings from the center of the front half and the center of the
back half of the tank.
Evaporated pheromone and skin lipid samples were exposed to either fullspectrum only or full-spectrum and UVB light for a total of 80 hours. For both the
6
pheromone and the skin lipid experiments, the samples categorized as “UV(+)” were
exposed to an average of 75.47 μW/cm2 UVB radiation, while those categorized as
“UV(-)” were exposed to an average of 0.57 μW/cm2 UVB radiation. During the live
animal exposure, snakes were exposed for 8 hours per day for a total of 15 days, with a 2day break occurring in the middle of the experiment. The average UVB exposure for live
snakes categorized as “UV(+)” was 9.18 μW/cm2, while the UVB exposure of snakes
categorized as “UV(-)” averaged 0.09 μW/cm2. For the Mylar filter test, the average
level of UVB under the Mylar filter was 0.915 μW/cm2, while that under the acetate was
4.98 μW/cm2.
Isolated Pheromone Experiment
Pheromone samples that had been collected and purified in a previous study were
used for this experiment. Samples in solution in hexane from 10 female snakes and 10
male snakes from the study were divided in half according to total mass of pheromone
and were allowed to evaporate in the bottoms of glass beakers of 250 ml average volume.
Half of each evaporated sample was placed under full-spectrum and UVB light, while the
other half was exposed to full-spectrum light only. After exposure, each half of each
sample was resuspended to a concentration of 1 μg/mL in hexane and analyzed by gas
chromatography.
7
Neat Solution Experiment
Sheds from 12 snakes (5 males and 7 females) were collected within 24 hours of
being shed and were soaked in hexane for 16 hours. This procedure was developed by
former student Elliot Finn, and yields skin lipids in lower concentration than from live
snakes but with the same proportions to each other. The solution containing all skin
lipids extracted by the hexane, hereafter referred to as the “neat solution,” was dried
under reduced pressure by a rotoevaporator at 35º C and weighed to obtain the total lipid
mass. Each sample was resuspended in hexane and divided in half by mass into glass
beakers of 250 ml average volume. As before, all were allowed to evaporate, and half of
each sample was exposed to full-spectrum and UVB light while the other was exposed to
full-spectrum only. After exposure, each half of each sample was resuspended to a
concentration of 1 μg/mL in hexane. These were divided in half again: one part to be
analyzed for all neat solution components by gas chromatography and mass spectrometry,
and the other part to undergo column chromatography to isolate squalene and the
pheromone, which was then evaporated, weighed, resuspended, and also analyzed by gas
chromatography. The prominent skin lipids analyzed, aside from the pheromone and
squalene, were oleic acid, octadecanoic acid, cholestadiene, cholestenol, cholestanol,
cholestanone, campesterol, and cholestenone.
Snake Care
Live snakes were captured from the Inwood den in the Interlake Region of
Manitoba, Canada, and transported to Oregon State University in May of 2010. Snakes
8
were fed ad libidum once per week, alternating between worms and small salmon fry, and
provided water ad libidum for drinking and soaking. Each snake was weighed before,
after, and once per week during the experiment. Snakes were euthanized using an
overdose of 1% Brevital sodium (0.0052 ml per gram of body mass).
At the onset of the exposure period, 24 males and 24 females were randomly
sorted into either the UV(+) or the UV(-) treatment groups. Snakes showing signs of
shedding were allowed to shed before beginning exposure, to ensure that as few snakes as
possible shed after exposure had begun. This resulted in two sets of snakes with different
timelines for exposure and behavior data, with the second group beginning and ending
exposure later than the first and a seven-day overlapping interval where all snakes were
being exposed simultaneously.
Snakes were kept in 10 gallon glass tanks in an
environmental growth chamber while not in the UV room. Tanks were furnished with
paper bedding, a rock for shedding, a cardboard tube, and half of a cardboard egg carton.
Males and females were kept separate at all times. The number of blemishes or lesions in
the skin was counted on each when the experiment ended. All snakes were euthanized at
the end of the exposure period and had their skin lipids extracted.
Behavior Studies
To determine whether red-sided garter snakes could detect and respond to UVB
radiation, two tests were done. First, three cardboard tubes were placed in each tank
during exposure for one hour. At the end of the hour, the number of snakes from each
experimental group seeking shelter within the cardboard tubes vs. being outside of them
9
was counted. The second test was based on a similar experiment done with tadpoles
(Belden et. al., 2003). Each tank being exposed to UVB light had a Mylar filter, which
filters out UVB light, placed over one half while an acetate sheet, as a control, was placed
over the other half. The Mylar filter was alternated between the front and the back halves
of the tanks to account for snake preference of a particular side of the tank. After one
hour with the filters on, the number of snakes situated under the Mylar vs. under the
acetate was counted.
For long-term effects of UVB radiation on snake behavior, snakes were observed
for a few minutes one hour before the end of the exposure time every day. The behaviors
that the snakes in each experimental group were displaying at that moment were
recorded. These behaviors were categorized as one of three types: active (crawling or
shifting position), alert (head up, tongue-flicking, not otherwise moving), and resting
(remaining motionless with head resting on a surface).
The percentage of snakes
displaying each behavior type was compared between experimental groups each day, as
well as over several different days in the same experimental group. Behavior surveys
were taken during two different time periods: the first seven days of exposure with the
first set of snakes, and the last seven days with the second set of snakes. No data was
used from the overlapping seven days because snakes with two different lengths of
exposure were present simultaneously.
Live-exposure Lipid Samples
Skin lipids were extracted from the snakes immediately after euthanasia by
soaking in hexane for 12 hours. Each was placed dorsal-side down in a glass jar filled
10
with hexane, with their heads and cloacas propped out of the liquid to avoid
contamination of the skin lipid sample with other body fluids. The extracts were dried
under reduced pressure on a rotoevaporator and weighed, then resuspended in hexane to a
concentration of 1 mg of lipid per 1 mL solution. Neat solutions from the sheds and live
animal exposure were divided in half, using one half to purify the pheromone and
squalene, and keeping the other half for direct gas chromatography/mass spectrometry
analysis of the other skin lipids in the samples.
Column Chromatography
Following the procedure described in Mason et. al, 1989, pheromone and
squalene samples were purified from the neat solution by fractionation on an alumina
activity III column, using glass columns 350 mm long by 11 mm in diameter. A mobile
phase consisting of pure hexane was used for fractions 1-3, which yielded squalene.
Fractions 4-6 were run using a mobile phase consisting of 98% hexane and 2% ethyl
ether, and contained the female sexual attractiveness pheromone.
Afterwards, each
sample was evaporated under reduced pressure on a rotoevaporator and weighed.
Gas Chromatography/Mass Spectrometry
The concentrations of the individual methyl ketones within the pheromone blend,
as well as squalene and the other skin lipids examined, were analyzed using a HewlettPackard 5890 Series II gas chromatograph fitted with a split injector (280ºC) and a
Hewlett Packard 5971 Series mass selective detector. Aliquots of 1 µL of the lipid
11
sample, along with 0.5 µL of a methyl stearate standard (20 µg/mL for directly exposed
pheromone and neat solution samples, plus pheromones from snakes from the first phase;
10 µg/mL for all other samples) and 1 µL of a hexane solvent, were injected into a fusedsilica capillary column (HP-1; 12 m by 0.22 mm in diameter; Hewlett-Packard) with
helium as the carrier gas (5 cm/sec). For each sample run, the oven temperature began at
70º C for 1 minute, then increased to 210º C at 30º C/min, was held at 210º C for 1
minute, increased to 310º C at 5º C/min, and held at 310º C for 5 minutes at the end of the
run. Compounds were identified and peak areas integrated using ChemStation software
(Version B.02.05, Hewlett Packard) on a computer interfaced with the gas
chromatograph/mass spectrometer.
Data Analysis
Compound concentrations were determined by integrating their peak areas and
calculating their proportions to the peak area of the methyl stearate standard of known
concentration.
Statistical analysis was done on data sets by performing one-way
ANOVAs and two-way repeated measures ANOVAs as appropriate.
conducted using SigmaStat software (Version 3.5; Systat Software, Inc.).
Tests were
12
Results
Isolated Pheromone Experiment
Comparisons between gas chromatograms of UVB-exposed pheromones and their
UV(-) counterparts from the same sample show distinct differences between the methyl
ketone profiles of each (Figures 2 and 3), most noticeably a reduction in the total methyl
ketone concentration in those exposed to UVB.
Figure 2. Gas chromatogram of an exemplary female pheromone sample exposed to
full-spectrum light for 80 hours.
13
Figure 3. Gas chromatogram of the same exemplary female pheromone sample as in
Figure 2, this time exposed to full-spectrum and UVB light for 80 hours.
Overall between the female sample experimental groups, seven out of eighteen of
the individual methyl ketones were significantly reduced in the UV(+) group from the
UV(-) group (Figure 4): those of molecular weight (Da) 476 (p0.001), 478 (p=0.002),
490 (p=0.013), 504 (p0.001), 506 (p=0.012), 518 (p0.001), and 532 (p0.001). The
US:S ratio was reduced (Figure 5.A) from 3.83 ( 1.51) in UV(-) samples to 2.10 (
1.11) in UV(+) samples (p=0.00934). In addition, the HMW:LMW ratio was reduced
(Figure 5.B) from 7.64 ( 3.24) in UV(-) samples to 2.15 ( 0.55) in UV(+) samples
(p=0.0000510). This is particularly demonstrative of the effects of UVB radiation on the
pheromone as both experimental groups began identically.
14
Figure 4. Average concentrations in solution of the 18 individual methyl ketones in
female isolated pheromone samples. Significant decreases in concentration in samples
exposed to UVB and full-spectrum light (UV+) from those exposed only to full-spectrum
light (UV-) for 80 hours were found in methyl ketones of molecular weight (Da) 476
(p0.001), 478 (p=0.002), 490 (p=0.013), 504 (p0.001), 506 (p=0.012), 518 (p0.001),
and 532 (p0.001).
15
Figure 5. A) Unsaturated:saturated methyl ketone ratios in female isolated pheromone
samples. A significant decrease in the ratio was found in samples exposed to UVB and
full-spectrum light (UV+) from those exposed only to full-spectrum light (UV-) for 80
hours (p=0.009). B) High molecular weight:low molecular weight methyl ketone ratios
in female isolated pheromone samples. A significant decrease in the ratio was found in
samples exposed to UVB and full-spectrum light (UV+) from those exposed only to fullspectrum light (UV-) for 80 hours (p=0.001).
16
Differences that arose in the male samples were similar to those of the females,
although less pronounced. Three of the eighteen methyl ketones were significantly
reduced by UVB exposure (Figure 6): molecular weights (Da) 476 (p=0.001), 504
(p0.001), and 532 (p=0.003). The HMW:LMW ratio was reduced from 5.75 ( 3.86) in
UV(-) samples to 2.01 ( 0.87) in UV(+) samples (p=0.023), while the US:S ratio was
reduced from 4.11 ( 1.31) in UV(-) samples to 2.88 ( 1.80) in UV(+) samples, although
not significantly.
Figure 6. Average concentrations in solution of the 18 individual methyl ketones in male
isolated pheromone samples. Significant decreases in concentration in samples exposed
to UVB and full-spectrum light (UV+) from those exposed only to full-spectrum light
(UV-) for 80 hours were found in methyl ketones of molecular weight (Da) 476
(p=0.001), 504 (p0.001), and 532 (p=0.003).
17
Neat Solution Experiment
Gas chromatograms of the neat solutions obtained from sheds show similar results
to the isolated pheromone experiment, although to a lesser degree (Figures 7 and 8).
Methyl ketones of molecular weight (Da) 476 (p0.001) and 504 (p0.001) in females
(Figure 9) and 504 (p=0.047) in males (Figure 11) were significantly reduced following
UVB exposure. The female US:S ratio was reduced (Figure 10.A) from 3.73 ( 1.42) to
2.04 ( 1.10) and the HMW:LMW was reduced (Figure 10.B) from 6.25 ( 2.45) to 3.76
( 1.50), both a lesser reduction than in the isolated pheromone experiment but still both
significantly (p=0.028 and p=0.041, respectively). The male HMW:LMW was reduced
from 7.39 ( 3.56) to 4.41 ( 1.65) while the male US:S ratio was reduced from 5.54
( 3.65) to 2.50 ( 1.27), although both not significantly.
18
Figure 7. Gas chromatogram of an exemplary male pheromone sample exposed to fullspectrum light for 80 hours.
Figure 8. Gas chromatogram of the exemplary male pheromone purified from the other
half of the neat solution in Figure 10, after exposure to full-spectrum and UVB light for
80 hours.
19
Figure 9. Average concentrations in solution of the 18 individual methyl ketones in
female skin lipid samples. Significant decreases in concentration in samples exposed to
UVB and full-spectrum light (UV+) from those exposed only to full-spectrum light
(UV-) for 80 hours were found in methyl ketones of molecular weight (Da) 476
(p0.001) and 504 (p0.001).
20
Figure 10. A) Unsaturated:saturated methyl ketone ratios in female skin lipid samples.
A significant decrease in the ratio was found in samples exposed to UVB and fullspectrum light (UV+) from those exposed only to full-spectrum light (UV-) for 80 hours
(p=0.028). B) High molecular weight:low molecular weight methyl ketone ratios in
female skin lipid samples. A significant decrease in the ratio was found in samples
exposed to UVB and full-spectrum light (UV+) from those exposed only to full-spectrum
light (UV-) for 80 hours (p=0.041).
21
Figure 11. Average concentrations in solution of the 18 individual methyl ketones in
male skin lipid samples. Significant decreases in concentration in samples exposed to
UVB and full-spectrum light (UV+) from those exposed only to full-spectrum light
(UV-) for 80 hours were found in the methyl ketone of molecular weight (Da) 504
(p=0.047).
No significant differences were found in the levels of squalene between the
treatment groups. Gas chromatography/mass spectrometry analysis of the other major
skin lipids revealed a decrease in the concentration of cholestenol (Figure 12) in male
samples (p0.001). The remaining lipids in the male samples (Figure 12), as well as all
of those in the female samples (Figure 13), showed no differences.
22
Figure 12. Average concentrations in solution of the major lipids in female skin lipid
samples exposed to either full-spectrum light only (UV-) or full-spectrum and UVB light
(UV+) for 80 hours.
23
Figure 13. Average concentrations in solution of the major lipids in male skin lipid
samples. A significant decrease in the concentration of cholestenol was found in samples
exposed to UVB and full-spectrum light (UV+) from those exposed only to full-spectrum
light (UV-) for 80 hours (p0.001).
Behavior Studies
No significant differences were found in either of the short-term laboratory
behavior tests. After one hour with the cardboard tubes, similar percentages of snakes
from both the “UV(+)” group and the “UV(-)“ groups were found seeking refuge within
the tubes as opposed to remaining outside of the tubes. At the end of the Mylar filter test,
24
no differences were found between the number of snakes choosing to place themselves
beneath the Mylar filter and the number of snakes counted beneath the acetate filter.
Over the length of the experiment, the percentages of snakes from both
experimental groups engaging in active, alert, or resting behaviors when observed held
mostly constant (Figures 14 and 15). There were also no differences in the percentages
of snakes engaging in each behavior between the two groups on any given day (Figures
14 and 15).
Figure 14. Percentage of snakes in each treatment group (exposed to full-spectrum light
only [UV-] or to full-spectrum and UVB light [UV+]) participating in one of three
behavior types (active, alert, or resting) each day for the first 7 days of exposure.
25
Figure 15. Percentage of snakes in each treatment group (exposed to full-spectrum light
only [UV-] or to full-spectrum and UVB light [UV+]) participating in one of three
behavior types (active, alert, or resting) each day for the last 7 days of exposure.
Live-exposure Lipid Samples
Despite the random sorting of snakes between the treatment groups, a size
difference occurred between the groups. The average final mass for female snakes (
standard deviation) in the UV(+) group was 79.15 g ( 20.17 g), while female snakes in
the UV(-) group averaged 62.08 g ( 13.36 g). This may have skewed the results towards
higher US:S and HMW:LMW ratios in the female UV(+) group, as snakes of higher mass
tend to produce pheromone blends with higher US:S and HMW:LMW ratios (Lemaster
and Mason, 2002). Male masses, and snout-vent length and mid-body circumference
between the treatment groups for both of the sexes were not statistically different.
26
Among the females, the skin surface concentration of the two most prevalent
individual methyl ketones, both unsaturated, were significantly lower in the UV(+)
treatment group than in the UV(-) treatment group (Figure 16). These were methyl
ketones of molecular weight (Da) 476 (p=0.002) and 504 (p0.001). In addition, the
US:S ratio was decreased (Figure 17) from 8.25 ( 3.08) to 5.57 ( 1.73, p=0.015). The
female HMW:LMW ratio showed no significant differences.
Figure 16. Average skin surface concentrations of the 18 individual methyl ketones in
live-exposure female pheromone samples. Significant decreases in concentration in
samples exposed to UVB and full-spectrum light (UV+) from those exposed only to fullspectrum light (UV-) for 8 hours per day for 15 days were found in methyl ketones of
molecular weight (Da) 476 (p=0.002) and 504 (p0.001).
27
Figure 17. Unsaturated:saturated methyl ketone ratios in the skin lipids of female
snakes. A significant decrease in the ratio was found in snakes exposed to UVB and fullspectrum light (UV+) from those exposed only to full-spectrum light (UV-) for 8 hours
per day for 15 days (p=0.015).
Among the other skin lipids analyzed in the female snakes, a significant increase
in concentration per square centimeter of skin was found in cholestenol (p0.001), from
6.27 g/cm2 ( 3.11 g/cm2) to 8.56 g/cm2 ( 4.09 g/cm2). This could be due to the
larger size of the females in the UV(+) treatment group, or to an un-regulation of the
production of protective skin lipids in response to UVB radiation. The differences in the
rest were insignificant (Figure 18).
28
Figure 18. Average skin surface concentrations of the major lipids in live-exposure skin
lipid samples from female snakes. A significant increase in the concentration of
cholestenol was found in samples exposed to UVB and full-spectrum light (UV+) from
those exposed only to full-spectrum light (UV-) for 8 hours per day for 15 days
(p0.001).
In the live animal exposure, no significant differences were found in any of the
methyl ketone concentrations (Figure 19) nor in either the US:S or the HMW:LMW
ratios between the male snake treatment groups.
29
Figure 19. Average skin surface concentrations of the 18 individual methyl ketones in
live-exposure male pheromone samples from snakes exposed to either full-spectrum light
only (UV-) or to full-spectrum and UVB light (UV+) for 8 hours per day for 15 days.
No significant differences in the concentrations of squalene were found between
the treatment groups. None were also found in the concentrations of any of the other
major skin lipids from the live-exposed male snakes. In addition, no differences in the
condition of the skin were found in either males or females between the treatment groups.
30
Figure 20. Average skin surface concentrations of the major lipids in skin lipid samples
from male snakes exposed to either full-spectrum light only (UV-) or full-spectrum and
UVB light (UV+) for 8 hours per day for 15 days.
31
Discussion
It is clear from the results of the exposure of the isolated pheromone that UVB
radiation does have a direct effect on the methyl ketones themselves, in particular, those
of long carbon chain length or that are unsaturated. Exposure of the pheromones, from
both females and males, when in solution with the other skin lipids yields a lesser change
in the pheromone concentration and US:S and HMW:LMW ratios, indicating that the
pheromone is still affected by UVB radiation when in a more natural mixture, but that the
other skin lipids have a shielding effect on the pheromone. The concentration change
found in cholestenol suggests that some of the other lipids may be absorbing part of the
radiation and being damaged as a result.
The behavior studies during the live-exposure period show no short- or long-term
behavior changes in the snakes due to exposure to UVB light. This indicates that redsided garter snakes either cannot detect UVB radiation or have no behavioral response to
protect themselves from it.
UVB levels during the live-animal exposure were similar to ambient levels that
occur naturally in the environment. The amount of UVB exposure experienced by each
snake over the 15-day exposure period would be close to that experienced by snakes in
the field within a couple of weeks after emerging from hibernation in the spring. The
changes found to occur in the pheromone during the time of the study are therefore likely
to be similar to those that occur in wild snakes during the breeding period. As the two
most prevalent of the methyl ketones in the pheromone profile decreased in
concentration, and the US:S ratio, known to be associated with the attractiveness of a
32
female snake (LeMaster and Mason, 2002), was also reduced in female snakes, exposure
to natural levels of UVB radiation may decrease the attractiveness of females over time
and have a profound impact on the breeding biology of these snakes in the field.
A serious concern raised by this study is the implication of an increase in UVB
radiation on the mating behavior of red-sided garter snakes in the field.
As UVB
radiation has been increasing along with ozone depletion, especially in the polar regions
of the globe such as the red-sided garter snake’s natural range (Kerr and McElroy, 1993),
the effects that ambient UVB has on the female sexual attractiveness pheromone may
become more profound. If UVB levels continue to rise, an impact on the attractiveness
of female snakes may be seen, with a potential shortening of the spring breeding season.
33
References
1. Bancroft, Betsy A.; Baker, Nick J. and Blaustein, Andrew R. (2007). Effects of
UVB radiation on marine and freshwater organisms: a synthesis through metaanalysis. Ecology Letters, 10: 332-345.
2. Belden, L. K.; Moore, I. T.; Mason, R. T.; Wingfield, J. C. and Blaustein, A. R.
(2003). Survival, the hormonal stress response and UV-B avoidance in Cascades
Frog tadpoles (Rana cascadae) exposed to UV-B radiation. Journal of Functional
Ecology, 17: 409-416.
3. Boch, R.; Shearer D. A. and Stone, B. C. (1962). Identification of iso-amyl
acetate as an active component in the sting pheromone of the honey bee. Nature
195: 1018-1020.
4. Butler, C. G. (1967). Insect Pheromones. Biological Reviews 42(1): 42-84.
5. Chang, Cheng and Zheng, Rongliang. (2003). Effects of ultraviolet B on
epidermal morphology, shedding, lipid peroxide, and antioxidant enzymes in
Cope’s rat snake (Elaphae taeniura).
Journal of Photochemistry and
Photobiology B: Biology, 72: 79-85.
6. Conant, R. (1975). A Field Guide to Reptiles and Amphibians: Eastern/Central
North America. 2nd ed. Houghton Mifflin, Boston.
7. Darwin, Charles. (1871). The descent of man, and selection in relation to sex.
John Murray, London.
8. Dennis, K. Jason and Shibamoto, Takayuki.
(1989).
Production of
malonaldehyde from squalene, a major skin surface lipid, during UV-irradiation.
Photochemistry and Photobiology 49(5): 711-716.
9. Dulka, J. G.; Stacey, N. E.; Sorensen, P. W. and Van der Kraak, G. J. (1987). A
steroid sex pheromone synchronizes male-female spawning readiness in goldfish.
Nature 325: 251-253.
34
10. Garstka, W. R.; Camazine, B. and Crews, D. (1982). Interactions of behavior
and physiology during the annual mating cycle of the red-sided garter snake
(Thamnophis sirtalis parietalis). Herpetologica 38: 104-123.
11. Gregory, Patrick T. (1974). Patterns of spring emergence of the red-sided garter
snake (Thamnophis sirtalis parietalis) in the Interlake region of Manitoba.
Canadian Journal of Zoology 52: 1063-1069.
12. Karlson, P. and Lüscher, M. (1959). “Pheromones”: a new term for a class of
biologically active substances. Nature 183: 55-56.
13. Kerr, J. B. and McElroy, C. T. (1993). Evidence for large upward trends of
ultraviolet-B radiation linked to ozone depletion. Science 262(5136): 1032-1034.
14. Kiesecker, Joseph M.; Blaustein, Andrew R. and Belden, Lisa K. (2001).
Complex causes of amphibian population declines. Nature 410: 681-684.
15. LeMaster, Michael P. and Mason, Robert T. (2002). Variation in a female sexual
attractiveness pheromone controls male mate choice in garter snakes. Journal of
Chemical Ecology 28(6): 1269-1285.
16. Mason, Robert T. and Crews, David. (1985). Female mimicry in garter snakes.
Nature 316(6023): 59-60.
17. Mason, R. T.; Fales, H. M.; Jones, T. H.; Pannell, L. K.; Chinn, J. W. and Crews,
D. (1989). Sex pheromones in snakes. Science 245: 290-293.
18. Moore, B. P. (1966). Isolation of the scent-trail pheromone of an Australian
termite. Nature 211: 746-747.
19. Noble, G. K. (1937). Sense organs involved in the courtship of Storeria,
Thamnophis, and other snakes. Bulletin of the American Museum of Natural
History 73: 673-725.
20. Rasmussen, L. E. L.; Lee, Terry D.; Zhang, Aijun; Roelofs, Wendell L.; Daves
Jr., G. Doyle. (1997). Purification, identification, concentration, and bioactivity
of (Z)-7-dodecen-1-yl acetate: sex pheromone of the female Asian elephant,
Elephas maximus. Chemical Senses 22(4): 417-437.
35
21. Ries, Gerhard; Heller, Werner; Puchta, Holger; Sandermann, Heinrich; Seidlitz,
Herald K. and Hohn, Barbara. (2000). Elevated UV(-)B radiation reduces
genome stability in plants. Nature 406: 98-101.
22. Say, T. (1823). IN: Edwin James, account of an expedition from Pittsburgh to
the Rocky Mountains. (Long’s expedition to Rocky Mountains). Longman, Hurst,
Rees, Orme, and Brown, London.
23. Shorey, H. H.; Gaston, L. K. and Fukuto, T. R. (1964). Sex pheromones of
noctuid moths. 1. A quantitative bioassay for the sex pheromone of Trichoplusia
ni (Lepidoptera: Noctuidae). Journal of Economic Entomology 57(2): 252-254.
24. Uhrig, Emily J.; Lutterschmidt, D. I.; Mason, R. T. and LeMaster, M. P.
Pheromonal mediation of intraseasonal declines in the attractivity of female redsided garter snakes, Thamnophis sirtalis parietalis. Journal of Chemical Ecology
(in review).
25. Wabnitz, Paul A.; Bowie, John H.; Tyler, Micheal J.; Wallace, John C.; and
Smith, Ben P. (1999). Animal behaviour: aquatic sex pheromone from a male
tree frog. Nature 401: 444-445.