The effects of vomeronasal blockade on prey discrimination in rattlesnakes
Abstract- Rattlesnakes release their prey after delivering an envenomating strike, allowing the
prey to wander away. To recover their prey, rattlesnakes exhibit a high rate of tongue flicking
and side to side searching with the head. These behaviors have been labeled strike-induced
chemosensory searching (SICS). Studies investigating the bifid tongue of rattlesnakes suggest
that chemical information that aids in prey recovery is brought in through the tongue to the
vomeronasal system (VNS). Previous work has investigated rattlesnake’s ability to distinguish
between prey envenomated by a conspecific rattlesnake and prey manually killed by the
experimenter and not envenomated. The purpose of the current study was to assess the
importance of the VNS in mediating a rattlesnake’s ability to distinguish between envenomated
and nonenvenomated prey items. The VNS was temporarily blocked with Xylocaine ointment
and the rattlesnake was presented with two prey items, one envenomated and one manually
killed and not envenomated. Xylocaine effectively inhibited the rattlesnake's VNS, indicating
that the VNS plays a critical role in mediating prey discrimination and SICS.
Introduction:
Although the predatory habits of rattlesnakes include opportunistic elements, such as scavenging,
more commonly rattlesnakes are known for their ambushing tactics. Rattlesnakes that feed on
rodent prey will seek an area well trafficked by mice. Once a rodent wanders within striking
range, the rattlesnake will deliver one envenomating strike and then immediately release the prey
(Chiszar, Radcliffe, Byers, & Stoops, 1986; Kardong, 1986; O’Connell, Chiszar, & Smith, 1981,
O’Connell, Greenlee, Bacon & Chiszar, 1982). The rodent will wander away from the site of the
attack before the venom takes effect (Estep, Poole, Radcliffe, O’Connell, & Chiszar, 1981).
After a strike, the rattlesnake begins to exhibit a sustained high rate of tongue flicking
(RTF; Chiszar & Radcliffe, 1976) accompanied by side-to-side searching movements with the
head. Because these behaviors are dependent upon the delivery of a predatory strike (Golan,
Radcliffe, Miller, O’Connell, & Chiszar, 1982), they have been termed strike-induced
chemosensory searching (SICS; Chiszar, Radclife, & Scudder, 1977). SICS enables the
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rattlesnake to locate the trail left by the envenomated rodent and recover the prey (Chiszar,
Smith, & Hoge, 1982; Golan, et. al., 1982; O’Connell, et. al., 1982).
It is generally accepted that the tongue serves as the vehicle to deliver chemical cues to
the vomeronasal system (VNS) because the oral ducts leading to the vomeronasal organs lay in
the direct pathway of the tongue (Young, 1993). This conclusion has been supported by studies
showing that a partial or complete removal of the bifid tongue results in a pronounced reduction
in prey trailing abilities in garter snakes (Halpern & Kubie, 1980). The sustained high RTF
observed during SICS therefore suggests the utilization of the VNS (Chiszar, Radcliffe,
O’Connell, & Smith, 1982; Halpern & Kubie, 1980, 1984; Kubie & Halpern, 1979).
Indeed, a study performed by Alving and Kardong (1996) attempted to investigate the
importance of the VNS through surgical procedures. Transections in the vomeronasal nerve
were made in order to deprive subjects of the use of their vomeronasal organs. As a result, the
rattlesnake’s trailing ability was significantly diminished. If the vomeronasal nerve was
completely cut, no trailing behavior or swallowing occurred.
The study performed by Alving and Kardong (1996) was significant because it provided
evidence suggesting the importance of the VNS in crotaline predatory behavior. However their
technique was flawed. Cutting the vomeronasal nerve reduced normal predatory strike behaviors,
resulting in 50% to 100% of subjects failing to strike (Alving & Kardong, 1996). Because
cutting the vomeronasal nerve interfered with strike behavior, this technique was ineffective for
studying behaviors, such as SICS, which are believed to be dependent upon the predatory strike.
A new technique developed by Stark, Chiszar, and Smith (2006) allows vomeronasallymediated behaviors, such as SICS, to be studied more effectively. Snakes are presented with
mice that have been covered with a thin layer of Xylocaine ointment. When a snake strikes the
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prey item, the Xylocaine ointment is transferred into the snake’s mouth where it passes through
the oral ducts to the VNS (Stark, et. al., 2006). Xylocaine ointment (5% lidocaine ointment in a
petroleum-based medium) has a consistency similar to Vaseline and is used to topically
anesthetize mucous membranes in medical and veterinary practices. In a study using Xylocaine
to investigate reproductive behavior in the adder, it was determined that the onset latency is
approximately two to three minutes, with a duration of about two hours (Andrén, 1982).
Two studies (Chiszar, Walters, Urbaniak, Smith, & Mackessy, 1999 and Duvall, Scudder,
& Chiszar, 1980) have shown that rattlesnakes can successful distinguish between prey
envenomated by a conspecific rattlesnake and prey manually killed by the experimenter. The
purpose of this study was to investigate the importance of the VNS in mediating the ability of
rattlesnakes to distinguish between envenomated (E) and nonenvenomated (NE) prey using the
new technique developed by Stark, et.al.
Materials and Methods:
Subjects. Fourteen rattlesnakes were used as subjects for this study (4 Crotalus viridis viridis, 3
Crotalus atrox, 3 Crotalus viridis oreganus, 2 Crotalus viridis helleri, 1 Crotalus horridus
horridus, and 1 Crotalus oreganus lutosus). All snakes had been in captivity for at least three
years and had been accepting rodent prey (Mus musculus) on a fortnightly schedule. All the
snakes were housed individually in glass terraria (60cm x 30cm x 42cm) with paper floor
coverings, stainless steel water basins, and one large rock for cover. The room was kept at 26 ±
1º during photophase and 23 ± 1º during scotophase. These snakes had been used in previous
experiments involving the presentation of chemical stimuli, however no surgical manipulation
had been performed. These snakes were representative of long-term captive rattlesnakes. All
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procedures had been approved by the Western State Animal Welfare Committee and were in
accordance with the Departments of Psychology and Biology.
Procedure. During all experimental conditions, snakes were observed in their home cages. All
snakes were presented with one of the following experimental conditions: 1) a target mouse
coated in Xylocaine ointment, or 2) a target mouse coated in Vaseline ointment.
Preparation of Target Mice. Target mice and mice used for the discrimination task (see
below) were killed by cervical dislocation just prior to use. For mice used in condition 1,
Xylocaine ointment (5% Lidocaine in a petroleum based jelly, Astra Pharmaceutical Products,
Inc., Westborough, MA 01581) was applied to the dorsal surface of a mouse carcass with a
wooden tongue depressor, completely covering the carcass from the head to the base of the tail
with a thin layer of ointment. After the application of ointment, the carcass was placed on a
warming tray, ventral side down, allowing the Xylocaine ointment to melt slightly, ensuring the
entire surface was covered. Mice used in condition 2 were prepared in a similar manner,
however Vaseline ointment was used in the place of Xylocaine ointment.
Presentation of Mice. Target mouse carcasses were suspended via long forceps into the
terraria. Mice were presented approximately 10 cm in front of and slightly elevated above the
subjects’ head (the same procedure used during normal feeding schedules). Mice were presented
with the dorsal surface facing the snake to ensure that during the strike the snakes’ mouth
contacted the dorsal surface containing the experimental ointment. After a strike, the target
mouse was immediately removed from the terraria.
Discrimination Task. A study performed by Melcer and Chiszar (1989) has shown that
rattlesnakes can discriminate between prey items based on chemical cues obtained during a
strike. Therefore, to control for the possibility of odor factors related to the Xylocaine or
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Vaseline having an effect on the snakes’ ability to discriminate between the envenomated and
nonenvenomated rodents, the coated target mice were not used during the discrimination task.
This experiment used the methods developed by Duvall, Scudder, and Chiszar (1980). At
approximately the same time that the subject struck the target mouse, an uncoated mouse carcass
of similar size and weight to the target mouse was envenomated by a conspecific rattlesnake.
The mouse envenomated by the conspecific was placed within a wire mesh bag (8cm x 12cm).
A third, non-envenomated mouse carcass was placed in an identical mesh bag. Both mesh bags
were fastened to a wooden board and then lowered in the snake’s terraria.
Hand-held counters were used to record the number of tongue flicks the snake emitted for
a total of twenty minutes. Specifically tongue flicks directly above or below the mouse in the
mesh bags and tongue flicks directed into the openings in the wire mesh were recorded. In
addition, accumulated time spent investigating each carcass was recorded.
All snakes were tested under both conditions and the order of presentation was
randomized. Results were analyzed using two-way repeated measures ANOVA and post hoc
comparisons were performed using match pairs t tests.
Results:
Figure 1 presents the mean number of tongue flicks directed towards envenomated and nonenvenomated prey during Vaseline and Xylocaine conditions. ANOVA revealed a significant
interaction between the two experimental conditions, [F(1,13)=20.38, P<0.05]. Post-hoc
comparisons indicated that snakes directed significantly more tongue flicks towards
envenomated prey than nonenvenomated prey under the Vaseline condition (t (13) =3.327,
P<0.05). Additionally, snakes directed significantly more tongue flicks towards envenomated
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prey during the Vaseline condition than during the Xylocaine condition (t (13) = 3.106, P<
0.05). No other effects were significant.
The mean investigation time during the two experimental conditions is presented in
Figure 2. ANOVA revealed a significant main effect of the experimental condition Vaseline
versus Xylocaine [F(1,13)=6.05, P<0.05] as well as a significant main effect of envenomated
versus non-envenomated [F(1,13)=4.46, P<0.05]. The interaction between the two independent
variables was also significant [F(1,13)=5.26, P<0.05]. Post-hoc comparisons indicated that, as
with RTF, snakes spent significantly more time investigating envenomated prey than
nonenvenomated prey under Vaseline conditions (t=2.278, P<0.05).
Discussion:
Previous studies investigating discrimination have concluded that rattlesnakes prefer
envenomated prey to nonenvenomated prey (Chiszar, Walters, Urbaniak, Smith, & Mackessy,
1999; Duvall, Scudder, & Chiszar, 1980). This ability to distinguish between envenomated and
nonenvenomated prey could be explained by the evolution of chemosensory capabilities that
allow the rattlesnake to detect the presence of venom. Because adult rodents can wander a
considerable distance from the site of an attack (Estep, et al., 1981), chemosensory searching
capabilities would be favored by natural selection.
A study performed by Duvall, Scudder, and Chiszar (1980) found that rattlesnakes are
able to discriminate between envenomated and nonenvenomated rodents, even when the
envenomated rodent is struck by a conspecific. More importantly, Duvall, et al. concluded that
those snakes discriminated the envenomated mouse using venom-related chemical cues
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remaining on the rodent after the strike. The authors hypothesized that the preference for the
envenomated mouse was due to a VNS-mediated chemosensory investigation.
The results of the present study provide definitive evidence supporting the conclusion
that the VNS is paramount to a rattlesnake’s ability to detect chemical cues deposited during a
strike. Striking a Xylocaine-coated mouse significantly inhibited the rattlesnake’s ability to
distinguish between envenomated and non-envenomated mice compared with striking a
Vaseline-coated mouse. Our findings replicate the results of a study performed by Alving and
Kardong (1996) that showed that snakes with vomeronasal nerve transections had a significantly
lower RTF when compared with intact controls. Furthermore, the present study provides
conclusive evidence to support the hypothesis proposed by Stark et al. (2006) that Xylocaine acts
to effectively block the VNS, which is the critical system used by rattlesnakes for prey
identification and discrimination.
The effects of Xylocaine appeared to have no outward influence on the snake’s searching
behaviors compared with controls. Rattlesnakes under the influence of Xylocaine commenced
SICS and continued to search for the full 20 minute trial period. However, snakes with an
anesthetized VNS exhibited an important difference from control subjects. Xylocaine ointment
increased the latency to locate mice in the discrimination apparatus. Although this was not
directly measured in the present study, Stark et. al. (2006) showed that Xylocaine significantly
increased the amount of time necessary to locate envenomated prey. It was observed in the
present study that snakes with an anesthetized VNS would search over the discrimination
apparatus many times before pausing to investigate. A possible explanation for this behavior is
that Xylocaine completely inhibited the subject’s ability to register chemical cues that would
allow the rattlesnake to distinguish between envenomated and nonenvenomated prey.
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It is important to discuss the fact that rattlesnakes with an anesthetized VNS did
eventually locate the discrimination box, directing tongue flicks towards the paired carcasses.
Most likely, the snakes used sensory cues other than those mediated through the VNS to
discriminate the prey items from the environment. Halpern and Kubie (1984) have shown that
garter snakes use airborne odorants to detect prey. In the present study it is possible that
rattlesnakes were able to detect the rodents as prey using visual, thermal, or olfactory cues, but
were unable to discriminate between envenomated and nonenvenomated rodents because the
VNS was anesthetized.
Observations from the present study demonstrate that Xylocaine anesthetization is an
effective, yet temporary and noninvasive technique. The analgesic effects of Xylocaine
dissipated quickly; within hours snakes were able to feed normally, locating and ingesting prey.
The present study provides addition evidences that the VNS is the primary system mediating
prey discrimination in rattlesnakes. Although subjects were eventually able to locate prey, they
were unable to discriminate between envenomated and nonenvenomated rodents. These results
indicate that Xylocaine acts as an effective temporary vomeronasal blockade.
Future studies should focus on the uses of particular sensory systems during post-strike
behaviors in rattlesnakes. The use of Xylocaine as a temporary anesthesia could be expanded to
investigate the importance of the VNS in mediating trailing and swallowing behaviors in
rattlesnakes. It is important to establish what sensory systems a rattlesnake uses to follow a
chemical trail and ingest their prey. Additionally, it should be determined what sensory systems
are used to discrimination prey items from the environment. The exact importance of the
olfactory, thermal, and visual systems during the post-strike period could have important
implications for further crotaline research.
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Mean Number of Tongue Flicks
200
P<0.05
150
P<0.05
E
NE
100
50
0
Vaseline
Xylocaine
Figure 1: Mean number of tongue flicks directed towards E and NE prey during Vaseline and
Xylocaine experimental conditions.
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Mean Investigation Time
200
P<0.05
150
P<0.05
E
NE
100
50
0
Vaseline
Xylocaine
Figure 2: Mean time spent investigating E and NE prey during Vaseline and Xylocaine
experimental conditions.
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