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 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 vomeronasally-mediated 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 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 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 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 non-envenomated 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 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 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.
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.