Hanisch 1

Chemosensory Mechanisms of Preferential Prey Capture by the Black Rat Snake,

Pantherophis Obsoletus
Jonathan Hanisch
Biology 457, Department of Biology, University of Washington
Introduction
Chemosensory mechanisms have evolved over millions of years, helping to facilitate
many different types of interactions between organisms. The main components of these
mechanisms are chemosensory organs, which can distinguish and interpret a wide variety of
chemical signals from predators, prey, and members of the same species. For example, while
emitted pheromones are known to mediate specific, often reproductive events between
individuals of the same species; emitted kairomones are known to enable predator-prey
interactions between two different species (Wyatt, 2003). Together, chemosensory mechanisms
enable organisms to detect, integrate, and respond to myriad chemical stimulus in the
environment.
In snakes, both the olfactory system and vomeronasal system allow the detection of semivolatile chemicals (Miller & Gutzke, 1999). Olfactory detection occurs via interactions between
chemicals and the olfactory mucosa, and vomeronasal detection is facilitated by the vomeronasal
organswhich line the roof of the mouth (Kubie & Halpern, 1978). The vomeronasal organs are
thought to receive chemosensory input from the tongue as it flicks out to the environment,
catches chemicals in the air and on the ground, and moves back into the mouth where it comes
into contact with the vomeronasal organs (Burghardt & Pruitt, 1975; Kubie & Halpern, 1978;
Wilde, 1938).

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Using the exquisitely-designed snake chemosensory systems, experiments can be
performed to help understand and evaluate predator-prey interactions. Specifically, a current area
of research involves studying factors that affect a preys emitted chemical odors, and how these
odors contribute to predator sensing and successful prey capture. This issue is significant because
understanding certain factors and behaviors that both affect a preys chemical signal and cause
the signal to be either more or less preferential to predators helps to evaluate evolutionary
relationships between species.
Background
Previous studies have shown that the snake olfactory system and vomeronasal system
allow snakes to perceive chemicals for predatory and reproductive purposes (Halpern, 1979;
Kubie & Halpern, 1978; Miller & Gutzke, 1999; Waters, 1993). In particular, blocking the
vomeronasal ducts decreases a snakes ability to follow an odor trail (Waters, 1993). This leads
to the conclusion that tongue flicking behavior in snakes and the relationship to the vomeronasal
organs is a mechanism of chemotaxis (Waters, 1993).
Additional studies have shown that suturing the vomeronasal ducts shut prevents
recognition of semi-volatile chemicals and hinders a snakes ability to detect predators and
instigate courtship behavior (Kubie & Halpern, 1978; Miller & Gutzke, 1999). For example,
Kubie et al. found that while the severing of the olfactory nerves in male garter snakes did not
impact mating and courtship behavior, blocking the vomeronasal organs inhibited such behavior
(Kubie & Halpern, 1978).
Despite extensive research on snake chemosensation, questions still remain regarding the
character of chemical odors processed by both the olfactory system and vomeronasal system. For

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example, in a predator-prey relationship, do certain prey behaviors alter the chemicals that are
sensed by a snake predator? And if so, what sensory systems preferentially detect, integrate and
cause a behavioral response in the predator?
Rationale
To this end, we will examine the claim that specific prey behavior results in the
modification of semi-volatile chemicals produced, and that snakes can differentiate between the
chemical odors emitted by prey items of a specific species to facilitate preferential prey capture.
Specifically, can snakes differentiate between the chemical odors emitted by over-fed versus
normally-fed prey items? And if so, which chemosensory system (olfactory, vomeronasal, or
both) facilitates detection of these odors?
The exploration of these questions will increase the understanding of snake prey
detection mechanisms. Additionally, it will determine if prey behavior-mediated chemical odor
changes are detected by predators, and if these changes are beneficial to either the predator or the
prey. Ultimately, this study aims to shed light on the evolution of predator-prey relationships
through lenses of prey behavior and predator chemosensory mechanisms.
From these questions, we will evaluate the hypothesis that over-fed prey items produce
chemically distinct odors from normally-fed prey items, and that snake vomeronasal organmediated chemosensation leads to the preferential hunting and capture of over-fed prey items
versus normally-fed prey items. If confirmed, this hypothesis will show that certain prey
behaviors (such as over-feeding) are detrimental to prey survival. This will add to past research
by verifying the importance of snake vomeronasal organs in chemosensory processes such as
predation. Also, additional studies may examine how chemosensory mechanisms such as the

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vomeronasal organs evolved to exploit chemical odors produced during prey behaviors that
indicate vulnerability. If disconfirmed, this hypothesis will suggest that chemical odors produced
by differential prey behavior do not have an effect on preferential prey capture.
Method and Design
This experiment will be performed using the black rat snake, Pantherophis obsoletus, and
the white-footed mouse, Peromyscus leucopus. The black rat snake is commonly found in the
southeastern United States where it preys on many species of squirrels, birds, and mice
including the white-footed mouse (Weatherhead, 2002). Their relatively small size, large overall
population density, and predator profile make the black rat snake ideal for this study. Whitefooted mice, besides being a main prey item for the black rat snake, have been shown to produce
chemical extracts that elicit predatory responses from snakes (Clark, 2003). All snakes and mice
will be collected from the wild and acclimated to the laboratory environment for one month
before testing.
One month prior to experimental testing with black rat snakes and white-footed mice, the
white-footed mice will be randomly separated into over-fed and normally-fed groups of n=10.
The over-fed group will be given one allotment of food (approximately three tablespoons per
mouse) three times daily, and the normally-fed group will be given one allotment of food once a
day as a control. Mice will be individually separated to prevent food competition and mating
behavior. Food will consist of the normal diet of the white-footed mouse: nuts, seeds, and fruit
(Hansen & Batzli, 1978).
To determine the baseline composition and over-fed composition of chemical odors
emitted by the mice in both groups, gas chromatography-mass spectrometry (GC-MS) analysis

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will be performed before and after the one month feeding cycle. To accomplish this, hair/skin oil
extract will be collected from both groups of mice before and after the one month feeding cycle.
Following procedures outlined in Kundu et al., a dorsal patch of hair/skin will be gently scraped,
and hypotonic buffer solution will be added before GC-MS analysis (Kundu & Shin, 2006).
Positive GC-MS results that confirm the hypothesis will show increased or decreased levels of
certain semi-volatile/volatile chemicals (such as aldehydes or ketones) in the over-fed group
when compared to the normally-fed group. Two-tailed t-tests will be performed to determine
significance in certain chemical odor levels between the over-fed group and normally-fed group.
Following the white-footed mouse one month feeding cycle and GC-MS analysis, black
rat snakes will be randomly separated into three groups of n=10: olfactory ability blocked,
vomeronasal organs blocked, or both olfactory ability and vomeronasal ability intact as control.
Following successful methods of snake chemosensory organ blocking in literaturebilateral
olfactory nerve cuts will be performed on the first group, vomeronasal nerve cuts on the second
group, and control surgery (consisting of drilling a hole in the skull but not severing nerve fibers)
will be performed on the third group (Kubie & Halpern, 1978). Snakes will be anesthetized
during surgery, will be allowed to recover for 2 days before beginning testing, and will be
histologically examined after experimental testing to verify olfactory and vomeronasal nerve cuts
(Kubie & Halpern, 1978).
The actual experiment consisting of both mice and snakes will utilize an airtight Tmaze (Waters, 1993). The use of this device will allow air flow to be controlled through a
vacuum pump (Appendix A). This will let snakes choose between the chemical odors presented
by both groups of mice. For each experiment run, a snake from one of the three groups will be
placed at the beginning of the T-maze. At each end of each point of the T will be a caged

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mouse from either the over-fed group or normally-fed group. The snake will be given 2 minutes
to acclimate to the maze, and then 10 minutes to move down the maze and choose between the
mice. Preferential black rat snake prey capture of over-fed versus normally-fed mice will be
based on the statistical analysis of how often snakes choose one group of mice over the other
while in the maze. A snake chooses between over-fed and normally-fed mice when more than
half of its body moves down the arm of the T-maze containing either mouse. If a snake does not
choose to move down either arm of the T-maze, it will be counted as no preference.
Experiments will occur at night between the hours of 2300-0100, as black rat snakes are
primarily nocturnal hunters (Amlaner, 1997). Mice from both groups will be assigned to
alternating arms of the T-maze to control for a snakes directional preference. Each snake from
the three snake groups will be placed in the T-maze with pairs of the 20 miceone over-fed and
one normally-fed. Therefore, each snake will be subjected to 10 experimental trials. Trials will be
recorded using infrared cameras in order to keep the experiment in a dark environment.
To ensure hunger and promote hunting behavior, snakes will not be fed until after
experimental trials are performed. Along these lines, snakes will run one trial per week to allow
full digestion of previous meals and to avoid the sedentary behavior that occurs during digestion
(Tattersall, 2003). Data will be compiled regarding individual snake preference to each mouse
pair. This data will then be compressed and analyzed using ANOVA statistical modeling.
Data showing that black rat snakes move to the end of the T-maze containing the over-fed
white-foot mice at a significantly higher frequency than the normally-fed white-foot mice will
confirm the hypothesis that snakes can detect and prefer behavior-induced pheromone changes in
prey (Appendix B). Conversely, data that disconfirm the hypothesis will show that no significant

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difference exists in black rat snake prey preference between over-fed mice and normally-fed
mice. Furthermore, data showing that snakes with severed olfactory nerves still preferentially
locate the over-fed prey at a higher frequency than snakes with severed vomeronasal nerves
supports the hypothesis that the vomeronasal organs mediate chemosensation of the chemical
odors emitted by the mice (Appendix B). If this is not the case and the olfactory system does
play a role in differential chemical odor chemosensation, then it is expected that snakes with
severed olfactory nerves will choose over-fed mice at a lower frequency than snakes with
severed vomeronasal nerves.
Significance and Conclusions
Overall, this research study aims to identify a difference in the chemical odor
composition of over-fed mice versus normally-fed mice and show that snakes can detect, and
preferentially prefer, the over-fed mice that emit this odor. This study relies on the assumption
that over-fed prey will provide a meal with higher energy content and will move more slowly
than their normally-fed counterpartsthus being easier to catch. There are many interspecific
examples in the natural environment where predators naturally deviate towards prey that are
weak, sick, or old in order to facilitate an easier kill (Cresswell & Quinn, 2004). Along these
lines, our prediction was that predators such as snakes can detect these changes through chemical
odors and integrate the information to consciously prefer disadvantaged prey.
Because current research has not identified specific attributes of prey behavior that cause
changes in chemical odor emittance and the effect on vertebrate predator-prey interactions, this
study represents the first-of-its-kind in the field. The identification of a specific prey behavior

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that changes the content of chemical odors emitted and the corresponding shift in prey preference
by the predator will have huge impacts on the field of evolutionary biology.
Specifically, how have predator-prey interactions evolved so that prey avoid behavior that
produces chemical odors preferred by predators? Also, how have predators evolved
chemosensory mechanisms to detect and prefer chemical odors that indicate prey vulnerability?
These are two of the future research questions that stem from this study.
Following experimental testing and data analysis, appropriate next steps include studying
other predator-prey systems at the invertebrate level to determine if the hypothesis is conserved.
Previous research on the interaction between mosquitoes and humans following beer
consumption will be a good starting point on which to build future experiments regarding
interspecific invertebrate trophic interactions (Lefevre & Gouagna, 2010).Other areas of future
research include analyzing how environmental pollutants change chemical odor emittance, and if
predator-prey relationships are impacted. Ultimately, understanding how behavior-induced
changes in chemical odor emittance lead to preferential prey capture is a vital issue in biology,
with implications in ecology and wildlife management, human and animal disease control, and
pollution control.

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Appendix A: Experimental Design, Air-tight T-Maze (Vacuum pump not shown)

1 meter
Wire cage
containing either
over-fed or
normally-fed
mouse

Wire cage
containing either
over-fed or
normally-fed
mouse

2 meters

Start

The T-maze is made out of black, opaque fiberglass with a transparent top to allow
infrared video recording. The maze is sealed to establish an airtight environment. Air-flow is
controlled through a vacuum pump, which prevents external contaminants from entering the
experimental area. The snake enters the maze at start and moves 2 meters down the maze
before choosing between an over-fed mouse and a normally-fed mouse.

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Appendix B: Potential Data that Confirm Hypothesis
1. (Below) Confirm hypothesis that over-fed mice emit unique chemical odors from
normally-fed mice, and that snakes can detect/prefer chemical odors of over-fed mice
using vomeronasal organs.
60
50
40
30
Abundance in sample

20
10
0

Normally-Fed
Over-Fed

Compound Classification per GC-MS

Figure 1: Over-fed mice exhibit elevated levels of aldehydes and ketones; and decreased levels
of benzenes compared to normally-fed mice in emitted chemical odor.

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90
80
70
60
50
% Trials Snakes Chose Over-Fed Mice

40
30
20
10

0
control (both nerves intact)
Experimental Treatment

Figure 2: Snakes with loss of vomeronasal nerve signaling choose

over-fed mice as prey significantly less than snakes with loss of
olfactory nerve signaling and control.

References
Amlaner, CJ. "Behavior Patterns in Free-Ranging Black Rat Snakes." NPWRC ::. N.p., n.d. Web. 16
Mar. 2015.
Burghardt, Gordon M., and Cheryl H. Pruitt. "Role of the Tongue and Senses in Feeding of Naive and
Experienced Garter Snakes." Physiology & Behavior 14.2 (1975): 185-94. Web.

Hanisch 12
Clark, Rulon W. "Timber Rattlesnakes (Crotalus Horridus) Use Chemical Cues to Select Ambush
Sites." Journal of Chemical Ecology 30.3 (2004): 607-17. Web.
Cresswell, W., and J. L. Quinn. "Faced with a Choice, Sparrowhawks More Often Attack the More
Vulnerable Prey Group." Oikos 104.1 (2004): 71-76. Web.
Daugherty, Laura A., and Nathan Pugh. "Effects of Snake Odor On the Behavior of Wild and Captive
Mice." Journal of Ecological Research 7 (2005): 64-67. Web.
Halpern, Mimi, and Natalie Frumin. "Roles of the Vomeronasal and Olfactory Systems in Prey Attack
and Feeding in Adult Garter Snakes." Physiology & Behavior 22.6 (1979): 1183-189. Web.
Hansen, Lonnie, and George O. Batzli. "The Influence of Food Availability on the White-footed
Mouse: Populations in Isolated Woodlots." Canadian Journal of Zoology 56.12 (1978): 2530541. Web.
Kubie, John L., Alice Vagvolgyi, and Mimi Halpern. "Roles of the Vomeronasal and Olfactory Systems
in Courtship Behavior of Male Garter Snakes." Journal of Comparative and Physiological
Psychology92.4 (1978): 627-41. Web.
Kundu, Joydeb, and Young Shin. "Resveratrol Inhibits Phorbol Ester-induced Expression of COX-2
and Activation of NF-B in Mouse Skin by Blocking IB Kinase Activity." Oxford University
Press 27.7 (2006): 1465-474. Web.
Lefvre, Thierry, Louis-Clment Gouagna, and Kounbobr Roch Dabir. "Beer Consumption Increases
Human Attractiveness to Malaria Mosquitoes." PLoS ONE 5.3 (2010): E9546. Web.
Miller, Lynda R., and William H.n. Gutzke. "The Role of the Vomeronasal Organ of Crotalines
(Reptilia: Serpentes: Viperidae) in Predator Detection." Animal Behaviour 58.1 (1999): 53-57.
Web.
Tattersall, G. J. "The Thermogenesis of Digestion in Rattlesnakes." Journal of Experimental Biology
207.4 (2004): 579-85. Web.
Waters, Roy Mark. "Odorized Air Current Trailing by Garter Snakes, Thamnophis Sirtalis." Brain,
Behavior and Evolution 41.3-5 (1993): 219-23. Web.
Weatherhead, P. J., G. Blouin-Demers, and K. A. Prior. "Synchronous Variation and Long-Term Trends
in Two Populations of Black Rat Snakes." Conservation Biology 16.6 (2002): 1602-608. Web.
Wilde, Walter S. "The Role of Jacobson's Organ in the Feeding Reaction of the Common Garter Snake,
Thamnophis Sirtalis Sirtalis (Linn.)." Journal of Experimental Zoology 77.3 (1938): 445-65.
Web.
Wyatt, Tristram D. Pheromones and Animal Behaviour: Communication by Smell and Taste.
Cambridge, UK: Cambridge UP, 2003. Print.