Effects of deprivation of vomeronasal chemoreception on prey discrimination in rattlesnakes.
Abstract: It has been demonstrated that rattlesnakes can discriminate between envenomed and nonenvenomed rodent prey based on venom-related cues deposited during the strike. This behavior is crucial to the snake's ability to choose the chemical trail left by an envenomed rodent fleeing the strike area and aids in the snake's ability to relocate the rodent The purpose of the current study was to assess the importance of the vomeronasal system (VNS) in mediating a rattlesnake's ability to distinguish between envenomed and nonenvenomed prey items. The VNS in adult rattlesnakes was temporarily blocked with Xylocaine ointment, and the rattlesnakes were presented with two prey items, one envenomated and one manually killed by the experimenter and not envenomed. Rattlesnakes in the Xylocaine condition failed to discriminate between envenomed and nonenvenomed prey items, whereas this discrimination was evident in the control condition. These data provide direct evidence that the VNS plays a critical role in mediating post-strike prey discrimination in rattlesnakes.

Key words: SICS, vomeronasal, rattlesnake
Article Type: Report
Subject: Rattlesnakes (Research)
Rattlesnakes (Physiological aspects)
Vomeronasal system (Research)
Vomeronasal system (Physiological aspects)
Authors: Stark, C. Patrick
Tiernan, Chelsea
Chiszar, David
Pub Date: 06/22/2011
Publication: Name: The Psychological Record Publisher: The Psychological Record Audience: Academic Format: Magazine/Journal Subject: Psychology and mental health Copyright: COPYRIGHT 2011 The Psychological Record ISSN: 0033-2933
Issue: Date: Summer, 2011 Source Volume: 61 Source Issue: 3
Topic: Event Code: 310 Science & research
Accession Number: 265302077
Full Text: Rattlesnakes exhibit several feeding strategies, including active foraging around rodent burrows and carrion eating. However, the modal predatory pattern involves waiting in ambush along trails frequented by rodents and delivering a single envenoming strike as soon as a rodent comes within range (Chiszar & Radcliffe, 1976; Clark, 2004, 2006; Dillcr, 1990; Klauber, 1972). After striking, the snake immediately releases the prey (within 0.3 s), and the rodent usually travels some distance from the site of envenomation before succumbing (Clark, 2006; Estep, Poole, Radcliffe, O'Connell, & Chiszar, 1981; Hayes & Galusha, 1984; Kardong, 1986). Within 3 to 8 min after the strike, the snake exhibits a high rate of tongue flicking and begins searching for the envenomed rodent (Clark, 2006; Golan, Radcliffe, Miller, O'Connell, & Chiszar, 1982; Kardong, 1986; Klauber, 1972).

It is widely assumed that there is a functional relationship in squamates between flicking of the tongue and delivery of chemical cues to the vomeronasal system (VNS; see Young, 1993), Electrophysiological data have demonstrated a close temporal correlation between the activity pattern of the tongue retractor system and stimulation of the sensory receptors in the vomeronasal organs (VNO; Meredith & Burghardt, 1978). Chemical cues delivered by the tongue to the VNO bind to receptors cells in the VNO lumen and stimulate axons in the vomeronasal nerve that project to the accessory olfactory bulbs (Jiang, Inouchi, Wang, & Halpern, 1990). Behavioral studies have suggested a direct relationship between tongue-flick rates and activity of the VNO (Graves & Halpern, 1989, 1990; Halpern & Kubie, 1980). Several aspects of viperid predatory behavior involve a high rate of tongue flicking (RTF), such as trailing of envenomed prey and discrimination of envenomed (E) prey from nonenvenomed (NE) prey (Bauman, 1927, 1928; Dullemeijer, 1961). It has been demonstrated that rattlesnakes can discriminate between envenomed and nonenvenomed rodents based on venom-related cues, a behavior that is crucial to the snake's ability to choose the chemical trail left by an envenomed rodent fleeing the strike area (Chiszar, Walters, Urbaniak, Smith, & Mackessy, 1999; Duvall, Scudder, & Chiszar, 1980). Both prey trailing and prey discrimination are behaviors that are believed to depend on the accurate detection and processing of prey-derived chemical information by the VNS (Burghardt, 1990; Chiszar, Radcliffe, & Scudder, 1977; Dullemeijer, 1961; Halpern, 1992). However, few studies directly assess the behavioral effects of vomeronasal deprivation on these behaviors. Transection of the vomeronasal nerves has been reported to affect the magnitude of the increase in RTF seen following a predcitory strike and to eliminate prey-trailing ability in rattlesnakes (Alving & Kardong, 1996). Removal of the tips of the bifid tongue to impede transport of chemicals to the vomeronasal epithelium has provided further, although indirect, evidence that VNS deprivation disrupts prey-trailing ability (Dullemeijer, 1961). There have been no attempts to assess the involvement of the VNS in the ability to discriminate between E and NE prey.

Most attempts to block VNS activity in rattlesnakes have dramatically affected early predatory behaviors such as the strike. Vomeronasal nerve transection has been shown to reduce the probability of eliciting a strike (Alving & Kardong, 1996), and suturing closed the oral ducts leading to the vomeronasal organs produced complete cessation of strikes (Graves & Duvall, 1985), making assessment of the effects of vomeronasal deprivation on poststrike behaviors difficult.

Therefore, the present study was designed to investigate the effect of VNS deprivation on prey discrimination using a reversible method of blocking vomeronasal activity that did not produce concomitant changes in strike behavior. Using the methods developed by Stark, Chiszar, and Smith (2006), we examined the effects of anesthetic blockade of the VNS on rattlesnakes' ability to discriminate between E and NE rodents.

Method

Subjects

Fourteen adult rattlesnakes were used as subjects (4 Crotalus viridis, 3 Crotalus atrox, 3 Crotalus oreganus, 2 Crotalus helleri, 1 Crotalus horridus, and 1 Crotalus lutosus). These snakes were housed individually in glass terraria (50 cm x 27.5 cm x 30 cm) containing paper floor coverings and stainless steel water basins. All snakes had been in captivity for at least 3 years prior to the start of this investigation and were accepting rodent prey (Mus musculus or Rattus norvegicus) on a fortnightly schedule. Although these snakes had been used in previous feeding experiments, no surgical or pharmacological manipulations were involved. Hence, the snakes were typical of long-term captives. The room was kept at 26 [degrees] C to 28 [degrees] C during photophase (0700-1900) and at 22 [degrees] C to 24 [degrees]C during scotophase. Snakes were deprived of food for 2 weeks prior to the start of testing.

Procedure

All snakes were observed in their home cages. A trial began with the subject being presented with either a target mouse carcass coated with Xylocaine ointment (experimental condition) or a target mouse carcass coated with Vaseline (control condition). Targets were suspended by forceps approximately 10 cm in front of and slightly above the snakes' heads such that the dorsal surface of the target was facing the snake. Within 1 to 3 s, the snakes oriented to these carcasses and struck and then released them. Following the strike, the mouse carcass was removed. At no time during the presentation of the mouse carcass was it allowed to touch any surface of the snake's cage.

Male mice (M. musculus, approximately 20 g) used for targets, as well as those used during the discrimination task (see below), were previously killed by cervical disconnect and stored frozen at -4 [degrees] C. On the day they were to be used, the mice were thawed to room temperature. Just prior to use in an experimental trial, they were warmed to approximately 35 [degrees] C on a warming tray placed in front of a standard electric space heater. Final temperatures were measured by a thermistor placed within the ear. Experimental target mice were treated in the following manner: Xylocaine ointment (5% Lidocaine in a petroleum-based jelly, Astra Pharmaceutical Products, Inc., Westborough, MA 01581) was applied to the dorsal surface of a warm carcass with a wooden tongue depressor such that the carcass was completely covered from the anterior to posterior regions in a thin coating of ointment. The carcass was then returned to the warming tray until body temperature returned to approximately 35 [degrees] C. This allowed the Xylocaine ointment to melt slightly, ensuring complete coverage of the dorsal surface. Control target mice were treated in the same manner, save that Vaseline ointment was used in the place of Xylocaine.

To control for the possibility that odor or some other factor related to Xylocaine or Vaseline would have an effect on the snake's ability to distinguish between the rodents, coated target mice were not used in the discrimination task. Using the methods of Duvall et al. (1980), an uncoated mouse carcass of a size and weight similar to the target mouse's was envenomed by a conspecific rattlesnake at approximately the same time as the target mouse was struck. This mouse was then placed within a wire mesh bag (5.7 cm x 12.7 cm). An NE mouse carcass with two pinpricks to simulate fang punctures was placed in an identical mesh bag. Both bags were fastened about 4 cm apart to a wooden platform (15 cm x 15 cm), which was then lowered into the snake's cage.

Handheld counters were used to record the number of tongue flicks the snake made toward each carcass for a period of 20 min. Specifically, tongue flicks made directly above or below the bag containing each mouse, and tongue flicks made directly into the openings in the wire mesh, were recorded. The accumulated time the snake spent investigating each carcass was also recorded. Each snake was tested under both conditions (Xylocaine vs. Vaseline) at the rate of one trial per week, and order of presentation of the conditions was randomized.

Dependent variables were analyzed by a 2 x 2 repeated measures A NOVA treating E versus NE mice and Vaseline versus Xylocaine as factors. No differences were noticed between the rattlesnake taxa on either dependent variable; in fact, all snakes exhibited similar results. Because the number of specimens of each species was small, inferential analyses were not applied to the taxon factor. Instead, data were pooled into a single sample (n = 14). Post hoc comparisons were performed using paired-comparison f tests.

Results

The total numbers of tongue flicks directed toward each mouse carcass (E vs. NE) are presented in Figure 1 as a function of the experimental condition (Xylocaine vs. Vaseline). An ANOVA revealed an interaction between E versus NE prey and Vaseline versus Xylocaine, F(l, 13) = 20.38, [rho] < .05. A post hoc comparison revealed that discrimination between carcasses occurred only during the Vaseline condition. Snakes directed a significantly greater number of tongue flicks toward E mice than NE mice during the Vaseline condition, t(13) = 3.33, [rho] <.01, whereas there was no significant difference in the number of tongue flicks directed toward E and NE mice during the Xylocaine condition, f(13) = 1.18, [rho] >.05. Additionally, snakes directed significantly more tongue flicks toward E prey during the Vaseline condition than during the Xylocaine condition, t(13) = 3.11, [rho] <.05.

[FIGURE 1 OMITTED]

The mean investigation time, mirroring the tongue-flick results, is presented in Figure 2. An ANOVA again revealed a significant interaction between the two independent variables, F(l, 13) = 5.26, [rho] <.05. Post hoc comparisons indicated that, as with RTF, snakes spent significantly more time investigating E prey than NE prey under the Vaseline conditions, t(13) = 2.28, [rho] < .05, but not during the Xylocaine condition, t(13) = 0.15, p >.05. Furthermore, snakes spent significantly more time investigating the E prey during the Vaseline condition than during the Xylocaine condition, t(13) = 2.93, [rho] <.05.

[FIGURE 2 OMITTED]

Discussion

Duvall et al. (1980) found that rattlesnakes discriminated between E and NE rodents, even when the E rodent was struck by a conspecific. Here we replicate those findings. Snakes during the Vaseline condition directed significantly more tongue flicks toward E mice than toward NE mice.

Duvall et al. (1980) concluded that snakes were cueing on venom-related chemical information deposited on or in the rodents' bodies during the strike. The behavioral index used in the study by Duvall et al. to measure the differential levels of investigation of the paired rodents was the rate of tongue flicking directed toward each carcass. The authors assumed that the observed tongue-flick behavior indicated that the VNS mediated chemosensory investigation. The results of the present study provide evidence supporting the conclusion that the VNS is crucial to a rattlesnake's ability to detect chemical cues associated with envenomation. Striking a Xylocaine-coated mouse eliminated the rattlesnakes' ability to distinguish between E and NE mice. Thus, the present study strongly supports the hypothesis that the VNS is the critical system used by rattlesnakes for discriminating E versus NE prey.

The effects of Xylocaine appeared to have little efferent influence on the snakes' searching behaviors compared with controls. Rattlesnakes in the Xylocaine condition commenced strike-induced chemosensory searching (SICS) and continued to search for the full 20-min 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 E prey. It was observed in the present study that although snakes with an anesthetized VNS flicked their tongues for the entire 20-min trial, they would pass over and around the discrimination apparatus many times before pausing to investigate either mouse, and the majority of lingual extrusions were aimed at places other than the discrimination apparatus. A possible explanation for this behavior is that Xylocaine completely inhibited the subject's VNS-mediated ability to register chemical cues that would allow the rattlesnake To distinguish between E and NE prey, or to recognize mice of any sort.

Rattlesnakes with an anesthetized VNS eventually located the discrimination apparatus, directing tongue flicks toward 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. Parker and Kardong (2005) have shown that while rattlesnakes prefer to use chemical trails deposited on the substrate to locate E prey, they can use airborne cues as well. A similar finding has been reported in garter snakes (Halpern & Kubie, 1984; Kubie & Halpern, 1979). In the present study, it is possible that the rattlesnakes were eventually able to detect the rodents using olfactory cues but were unable to discriminate between E and NE rodents because the VNS was anesthetized.

Observations from the present study demonstrate that Xylocaine anesthetization is an effective temporary and noninvasive technique. The analgesic effects of Xylocaine dissipated quickly; within hours the snakes were able to feed normally, locating and ingesting E prey.

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Preparation of this article was supported by funding provided by the Thornton Research Grants Program.

Correspondence concerning this article should be addressed to Charles Patrick Stark, Associate Professor of Psychology, 213 Kelley Hall, Dept. of Behavioral and Social Sciences, Western State College of Colorado, Gunnison, CO 81231. E-mail: pstark@western.edu

C. Patrick Stark Western State College

Chelsea Tiernan Western State College

David Chiszar University of Colorado, Boulder
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